Host specificity testing predicts Eadya daenerys (Hym.: Braconidae), a potential biological control agent for the invasive pest Paropsis charybdis will be host specific to Paropsini (Col.: Chrysomelidae: Chrysomelinae)

T.M. Withers1, C.L. Todoroki1 G.R. Allen2, A.R. Pugh1 and B.A. Gresham1

1Scion, Private Bag 3020, Rotorua 3046, New Zealand, 2 Tasmanian Institute of Agriculture, University of Tasmania, Private Bag 98, Hobart TAS 7001, Australia, Corresponding author: [email protected]

Abstract The introduction of a new biological control agent is being proposed in New Zealand for Paropsis charybdis (Coleoptera: Chrysomelidae: Chrysomelinae), a eucalypt pest of Australian origin. The Australian solitary larval endoparasitoid Eadya daenerys (Hymenoptera: Braconidae) has been the subject of host range testing against non-target species in containment. The results of testing against two pest paropsine beetles, one native species, and six beneficial biological control agents are reported. All non-target species were springtime-active, external leaf-feeding larvae. Physiological development through to emergence of the parasitoid larva from the host only occurred within species in the tribe Chrysomelini; that is, the target P. charybdis (30 - 34%) and another eucalypt pest Trachymela sloanei (12.5%). Unsuccessful internal parasitism by E. daenerys was discovered upon dissection of four other non-target Chrysomelinae, Dicranosterna semipunctata (1.6%), Allocharis nr tarsalis (7.5%), Chrysolina abchasica (1.8%) and Gonioctena olivaceae (5.2%). Although not a physiological host for the parasitoid, the attack on the endemic beetle larva A. nr tarsalis was concerning. Oviposition attack- insertions were occasionally observed in all non-target species in the petri-dish assays (mean 0 - 1.6 attacks/min) including A. nr tarsalis, however they were significantly less when compared to P. charybdis and T. sloanei (mean 1.1 – 4.4 attacks/min). The order of presentation had no significant impact on the propensity of E. daenerys to attack non- targets. Considering the low propensity to attack, the different feeding niche (shrubs unrelated to Eucalyptus) and the sub-alpine habitat (greater than 1200m a.s.l.) of New Zealand endemic beetles such as A. nr tarsalis, we conclude the likelihood that this parasitoid will encounter and cause mortality to A. nr tarsalis is very low. Our results are consistent with field host relationship studies in Tasmania, and in combination indicate that E. daenerys is highly unlikely to attack any species apart from pest paropsine (Chrysomelini) species feeding on Eucalyptus. Eadya daenerys is proposed as a safe parasitoid for release into the New Zealand environment.

Keywords: biological control, host specificity testing, Chrysomelidae, paropsine, beneficial, Allocharis tarsalis, weed biological control agent, eucalyptus pest.

INTRODUCTION Host range testing prior to the introduction of classical biological control agents is undertaken to determine the potential risk of negative impacts on non-target species in the receiving country (Barratt et al. 2006). The key to biological control safety is considered by many to be introducing only natural enemies with host specificity limited to the target pest (Sands 1997; Keller 1999). There is a consensus amongst arthropod biological control scientists that phylogeny is a valuable starting point for predicting and assessing the field host range of a parasitoid (Hoddle 2004), but that other criteria such as ecological similarities are also very important (Kuhlmann et al. 2006; van Lenteren et al. 2006). We used this now well-established approach to arthropod host specificity testing to draw up a priority list of non-targets against which to undertake host range testing of Eadya daenerys Ridenbaugh 2018 (Hymenoptera: Braconidae) (Withers et al. 2015; Withers et al. 2018). This parasitoid was previously identified as Eadya paropsidis Huddleston & Short (Hymenoptera: Braconidae). It is a solitary larval koinobiont parasitoid that targets Eucalyptus leaf beetles in the genera Paropsis and Paropsisterna (abbreviated to Pst.) (Coleoptera: Chrysomelidae: Chrysomelinae) in Australia (Tanton & Epila 1984; De Little 1989; Rice 2005a). This parasitoid has been studied in the field in shining gum Eucalyptus nitens (H. Deane & Maiden) Maiden plantations in Tasmania, Australia, where it was associated with Paropsisterna agricola (Chapius) in springtime, a pest of young plantation eucalypts (Rice 2005a; Rice & Allen 2009). Field research on host parasitoid interactions, along with molecular and taxonomic studies, has revealed a species complex, redescribing E. paropsidis and identifying the primary parasitoid of Pst. agricola as Eadya daenerys Ridenbaugh 2018 (Peixoto et al. 2018). To date, E. daenerys has only been recorded in Tasmania, where apart from parasitism of Pst. agricola, it also readily attacks Paropsis charybdis Stål, as well as Pst. bimaculata (Olivier 1807), and Pst. nobilitata (Erichson 1842) (Peixoto et al. 2018). Eadya daenerys is a large (ca. 10-12 mm) black braconid wasp with a bright orange head. Eadya approach host larvae and rapidly oviposit a small (0.3 mm), hydropic egg directly into the haemocoel (Rice 2005a). Eggs expand by absorbing water and nutrients from the host before hatching (Quicke 1997). Combined egg and larval development occurs in 25 days at 18°C within the host (Rice & Allen 2009). The E. daenerys larva emerges from the host’s prepupal stage within the soil, spins a brown silk cocoon, and then undergoes an obligate pupal diapause until the following summer (Rice 2005b). Eadya daenerys is univoltine in Tasmania, with adults present in November to January. The peak abundance of E. daenerys in December coincides with peak Pst. agricola egg laying (Rice 2005b). The Eucalyptus tortoise beetle, Paropsis charybdis, has been present in New Zealand since 1916 (Clark 1938; Styles 1970). P. charybdis continues to be the most significant defoliating pest of the adult foliage of Symphyomyrtus eucalypts in New Zealand (Bain & Kay 1989; Withers & Peters 2017). In particular, E. nitens pulp plantations throughout the country can be heavily defoliated (Murphy & Kay 2000), and numerous other Eucalyptus species being developed for a solid wood industry are also highly palatable to the pest (Lin et al. 2017). The cost of managing P. charybdis (Withers et al. 2013) is a risk for all new eucalypt forest plantations throughout New Zealand. Existing biological control agents that attack the egg stage of P. charybdis have proven to only be adequate in controlling the second summer generation of the pest (Murphy & Kay 2000; Mansfield et al. 2011). The first spring generation is inadequately controlled by the egg parasitoids (E. Peters, unpublished data). Eadya daenerys holds promise for decreasing survival of the spring generation of P. charybdis. Potential negative impacts upon non-target organisms constitute the most important factor in assessing the impact on the receiving environment (Barratt et al. 2010). Prior to initiating host range testing with E. daenerys, a thorough analysis was undertaken to establish the most appropriate non-target beetle species in New Zealand to test (Withers et al. 2015). Beetles were selected based on how closely related they were to the target P. charybdis. Tortoise beetles belong to the tribe Chrysomelini in the sub-family Chrysomelinae. The sub-familial relationships recommended by Reid (1995) and Leschen and Reid (2004) are that the sister sub-family to the Chrysomelinae are the Galerucinae, so we considered all species within these two subfamilies. New Zealand has over 40 native species of Chrysomelinae in 5 genera: Allocharis, Aphilon, Caccomolpus, Chalcolampra and Cyrtonogetus (Leschen & Reid 2004; Reid 2006). All of these species belong to the subtribe Phyllocharina, while Paropsis belongs to a separate subtribe; Paropsina (Reid 2006). New Zealand native Galerucinae are divided into two tribes: the Galerucini and the Alticini (Nadein & Bezděk 2014). All Galerucini are thought to have root-feeding larvae, based on the biology of closely-related species overseas. All Galerucini in New Zealand belong to the “rootworms” subtribe Luperina and no larvae have ever been collected, presumably as they are restricted to the soil (Samuelson 1973). The Alticini are more varied and all are very small (Samuelson 1973). After reviewing all literature and collection records (C. Wardhaugh et al, unpublished data), we identified the largest native species with the greatest likelihood of having leaf feeding larvae, and having a suitable phenology of being active in late spring or summer. Originally we had hoped to find larvae for host specificity testing of Chalcolampra speculifera Sharp (Withers et al. 2015), the largest native Chrysomeline beetle, but it now appears this species larva is nocturnal and lives within refugia (C. Wardhaugh et al, unpublished data). We undertook field work in Kahurangi National Park (Northwest Nelson, New Zealand) to try to locate C. speculifera or larger Allocharis spp. or Caccomolpus spp. (e.g., C. amplus Broun, 1921)(Withers et al. 2015). All species are classified as “naturally uncommon” (Leschen et al. 2012). We only succeeded in locating larvae for host testing of Allocharis nr. tarsalis Broun 1917 (identified by R. Leschen) one of the largest species that feeds externally on leaves of Veronica albicans (Pétrie) Cockayne (Plantaginaceae) (Wardhaugh et al. 2018). Host testing was undertaken with nine species that included native, introduced beneficial (weed biological control agents), and pest species (Withers et al. 2015) (Table 1 and Supplementary Data 1), to evaluate the potential risk of E. daenerys to non-target species.

Table 1. Host plant, biology and development parameters of non-target Chrysomelindae species tested against E. daenerys. Non- targets ordered left to right from most closely to least closely related phylogenetically to the target host. “?” indicates uncertainty.

Target Non-targets Common Eucalyptus Small tortoise Blackwood Veronica leaf Tutsan leaf Broom leaf Heather Alligator Tradescantia Green name tortoise beetle beetle tortoise beetle beetle beetle beetle beetle weed leaf beetle thistle leaf beetle beetle Sub-family Chrysomelinae Chrysomelinae Chrysomelinae Chrysomelinae Chrysomelinae Chrysomelinae Galerucinae Galerucinae Criocerinae Cassidinae

Tribe Chrysomelini Chrysomelini Chrysomelini Phyllocharitini Gonioctenini Gonioctenini Luperini Alticini - Cassidini

Genus Paropsis Trachymela Dicranosterna Allocharis Chrysolina Gonioctena Lochmaea Agasicles Neolema Cassida

Species charybdis sloanei semipunctata nr tarsalis abchasica olivacea suturalis hygrophila ogloblini rubiginosa

Status Exotic pest Exotic pest Exotic pest Native Beneficial weed Beneficial weed Beneficial Beneficial Beneficial Beneficial Naturally BCA BCA weed BCA weed BCA weed BCA weed BCA uncommon Host plant Eucalyptus Eucalyptus Acacia Veronica Hypericum Cytisus Calluna Alternanthe Tradescantia Cirsium spp. spp. melanoxylon albicans androsaemum scoparius vulgaris ra fluminensis arvense philoxeroide s Host family Myrtaceae Myrtaceae Mimosaceae Plantaginaceae Hypericaceae Fabaceae Ericaeae Amaranthac Asteraceae Asteraceae eae

Generations Bivoltine Bivoltine Univoltine Univoltine? Univoltine Univoltine Univoltine Bivoltine Bivoltine Univoltine p.a.

Larval ecology Diurnal leaf Nocturnal leaf Diurnal leaf Diurnal leaf Diurnal leaf Diurnal leaf Diurnal leaf Diurnal leaf Diurnal leaf Diurnal leaf feeder feeder feeder feeder feeder feeder feeder feeder feeder, feeder, faecal shield faecal shield

No. instars 4 4 4 4? 4 4 3 3 5 4

Mean pupal 153.4 38.4 91.2 22.1 26.0 11.5 11.2 7.7 7.5 19.7 fresh weight (mg) Mean days L1- 24 54 18 25-35? 50 20 18 17 15 18 pupa at 18˚C±2 METHODS Parasitoids Experiments were undertaken in 2014 and 2015 to establish a laboratory colony of E. daenerys in New Zealand; however, mortality was high in the laboratory due to incompletely-spun pupal cocoons and pupal diapause proved difficult to break. We could not rely on the laboratory colony to supply sufficient females for host testing so instead field collected adults of unknown age on the wing from Eu. nitens plantations in Tasmania (Moina forest 41°32'27"S 146°04'38"E, Runnymede Forest 42°38'08.9"S 147°33'57.9"E, Ellendale Forest 42°38'07.24"S 146°45'04.24"E) between 28 November, and 12 December 2014, 2015, 2016 and 2017. These forests had not been sprayed with alpha-cypermethrin to control leaf beetles since 2010. Adult female wasps were shipped in chilled boxes to the containment facility in Rotorua, New Zealand, within seven days of collection, carried in glass vials with mesh inserts in their lids, and provisioned with a male (sometimes) and a smear of sugar. They were maintained in a 14-18 ℃ temperature- controlled cabinet with a constant supply of honey for a maximum of 35 days before being used in experiments. In 2014 and 2015 they were maintained in glass vials at 18 ℃ but this reduced survival to 14-18 days. In 2016 and 2017 they were transferred to individual ventilated 500 ml plastic containers lined with a paper towel, moistened with a spray of water droplets every 48 hours and kept at 14 ℃ and 14:10 L:D. These conditions increased survival duration to 35 days.

Beetle colonies The P. charybdis colony was initiated from adults collected each November from various Eu. nitens forests in the central North Island of New Zealand, and maintained in the laboratory at 18 ℃ and 14:10 L:D in large ventilated Perspex cages (1.0m x 0.8m x 0.6m). The base was lined with paper towel and dead leaves and foliage. Twice per week adult beetles were given a freshly-cut unsprayed branch of Eu. nitens foliage bearing adult leaves and new flush, with their cut bases sitting in tap water. These foliage branches were stored for up to one week prior in a 4 ℃ chill room with their bases sitting in water, covered by a large plastic bag to reduce transpiration. Egg laying commenced in late November and egg batches were removed daily into individual petri dishes. As batches hatched, groups of 8-12 larvae were transferred onto cut flush foliage tips of Eu. nitens in 50 ml ventilated plastic sauce containers for 4-5 days before being used in experiments as second instar larvae. Non-target beetle colonies were similarly reared. The only notable differences in laboratory rearing techniques were: Trachymela sloanei (Blackburn) were maintained on a mix of Eu. leucoxylon subsp. leucoxylon F.Muell., and Eu. nitens foliage in the form of freshly cut branches. The colony originated from adults collected in December of 2016 and 2017 from amenity Eucalyptus trees. These adults were found sheltering beneath the bark during the day, in Napier city, Hawkes Bay, New Zealand. Larvae were transferred to containers separate from adults to mature until at the stage suitable for experiments (approximately second instar). Dicranosterna semipunctata (Chapuis) were maintained in 300ml plastic containers on small sections of Acacia melanoxylon R.Br foliage bearing fresh expanding phyllodes that were cut from the tree each morning, with the bases inserted into tap water or damp tissue. The colony originated from unhatched eggs or L1 larvae collected from trees growing at Scion, Rotorua, New Zealand. Allocharis nr tarsalis were maintained in 300ml ventilated plastic containers on small sections of potted V. albicans plants, with the base of the container covered in dampened sphagnum moss. The colony originated from L1-L3 larvae collected from Mt Arthur between 1200 and 1400m a.s.l., Kahurangi National Park, New Zealand (-41.1976 lat 172.7133 Long), collected under DoC permit 54216-RES. Chrysolina abchasica (Weise) were maintained in 4L ventilated plastic containers on numerous freshly cut sections of potted Hypericum androsaemum L. with the base of the container covered in dampened vermiculite. The colony originated from adults collected the year prior in Georgia (Europe), and transferred to Scion containment from the Landcare Research insect containment facility, Lincoln. Larvae were transferred to containers separate from adults to mature until at the stage suitable for experiments. Gonioctena olivacea were maintained in 4L ventilated plastic containers on potted Cytisus scoparius (L.) Link plants with the base of the container covered in damp filter paper. The colony originated from adults collected in spring (November 2015) from Tongariro National Park, New Zealand. Larvae were transferred to containers separate from adults, feeding on cut foliage to mature until at the stage suitable for experiments. Experimental larvae were reared in ventilated 50mL plastic vials with several sprigs of fresh C. scoparius, replaced twice weekly. Lochmaea suturalis (Förster) were maintained in 4L ventilated plastic containers on numerous freshly cut sections of Calluna vulgaris (L.) Hull with the bases sitting in damp florists foam. The colony originated from adults collected in spring from Tongariro National Park. Larvae were transferred to containers separate from adults to mature until at the stage suitable for experiments, and maintained on small cuttings replaced every 48 to 72 hours. Agasicles hygrophila Selman & Vogt were maintained in 1L unventilated plastic containers on numerous freshly cut sections of Alternanthera philoxeroides (Mart.) Griseb. The colony originated from adults collected in spring (November 2015) in Northland, New Zealand. Larvae were transferred to 200ml plastic containers separate from adults and maintained on small cuttings, replaced every 48 to 72 hours, to mature until at the stage suitable for experiments. Neolema ogloblini (Monrós) were maintained in a large soft mesh cage enclosing a potted plant (20 stems growing in a 5L pot) of Tradescantia fluminensis Velloso. The colony originated from adults from Landcare Research, Lincoln in 2015. Larvae were transferred to 200ml plastic containers separate from adults and maintained on small cuttings, replaced every 48 to 72 hours, to mature until at the stage suitable for experiments. Cassida rubiginosa Müller were maintained in 4L ventilated plastic containers on potted Cirsium arvense (L.) Scop. The colony originated from adults collected in spring (October 2016) in Masterton, Wairarapa. Egg batches were transferred to 300ml plastic containers separate from adults, with a fresh leaf provided every 48 hours, to mature until at the stage suitable for experiments on cut leaves.

Physiological Assays – 24 hours no-choice exposure Experimental arenas were upright 750ml plastic containers (174mm H x118mm W x58mm D), with a 4x4 cm ventilated hole cut in the lid, which was lined with nylon “nappy liner” to prevent insects escaping. A piece of paper towel dampened with distilled water was placed on the base of the container, and a smear of liquid honey was placed on the mesh liner to provide food to parasitoids. Eight larvae (generally second instar) of the non-target species were settled with a camel hair brush onto a 6-8 cm sprig of fresh foliage, and its base was placed into a soaked 1cm3 of “florists” phenolic foam. Female E. daenerys utilised in 24 hour physiological assays were chosen on the basis of having been observed undertaking successful attack-insertions towards P. charybdis larvae in behavioural assays earlier that day. This suggested they were in a suitable physiological state. One female was introduced to the cage and left for exactly 24 hours, after which it was removed. After each assay all larvae were reared as a group on their host plant in the ventilated 750ml plastic container in the same laboratory for up to four weeks, or until pupation, whichever came first. Fresh foliage was provided every few days as required. All larvae were transferred to clean containers weekly and at that time any premature mortality, successful pupation to beetle, or emergence of an E. daenerys larvae was recorded. Pupation was indicative of not having been successfully parasitized. Any larvae or prepupae that died prior to pupation were held until decomposition began, and at this point were labelled, and frozen individually. In order to ascertain whether larval death was attributable to parasitism, these dead larvae were thawed, and carefully examined under a dissecting microscope at increasing magnification; 8x, 25x and then 50x. Any parasitoid remains were noted, described in detail and photographed for future reference. We aimed to complete between 12 and 16 replicates on each species but in a few cases a low supply of larvae limited the number of replicates conducted (T. sloanei with 5 reps and A. nr tarsalis with 8). There were two divergences from the above method. Firstly, Agasicles hygrophila enter the hollow stems of their host plant to undergo prepupation and pupation. In order to avoid disturbing them and causing unintended mortality, stems were only dissected one week after all larvae had disappeared. Unfortunately this meant some larvae were never accounted for and no dead bodies were ever found. Secondly, during testing of D. semipunctata on short branches of its host plant, blackwood, A. melanoxylon, a double plastic arena system was used both during testing and to rear the larvae in order to maintain the phyllode vitality. Cuttings of blackwood foliage were inserted into an upper test chamber (395 ml) in each container, with stems inserted into water within a lower test chamber (535 ml) and held in place by a folded paper wick. The larvae were introduced to the foliage, and then a nylon liner was fitted to prevent the larvae from getting trapped under the ventilated lid lip. Some mortality of larvae occurred unexpectedly as a result of them either consuming single strands of the nylon liner or drowning in water that had wicked up from the lower test chamber into the upper test chamber.

Behavioural observations No-choice sequential tests Experimental arenas were large glass petri dishes measuring 140 mm diameter x 22mm high. Experiments were conducted under both fluorescent and natural lighting within a PC2 insect containment laboratory at 20℃ and 75% relative humidity in Rotorua, New Zealand. Eight second instar beetle larvae were settled onto a 5-8cm foliage tip or sprig of their host foliage, in clean petri dishes. For each replicate an independent (not used prior in the same assay type) female E. daenerys was introduced into one of the glass arenas, alternating first contact with either the target host (eight larval P. charybdis) for 10 minutes (A) or non-targets, then moved promptly on to the non-target host (B) in the B arena or vice versa, and observed for one consecutive 10-minute period. Behavioural observations began once the parasitoid contacted the leaf or foliage sprig. We aimed to complete a minimum of 12 replicates on each species. After each assay, larvae were reared and fates recorded as for the physiological assays. The only assays discarded were those in which a female never contacted or attacked a single P. charybdis.

Two-choice tests Experimental arenas were large glass petri dishes measuring 140 mm diameter x 44mm high with eight second instar beetle larvae on a 5-8cm foliage tip or sprig of their host foliage. Paropsis charybdis larvae were settled on a Eu. nitens sprig, and non-target larvae were settled on their appropriate non-target foliage sprig (Table 1). Recording began when the female wasp was introduced to the dish and first contacted either of the host plants. The same behaviours were recorded but analysed on the basis of which larval species the behaviour was directed towards, and/or upon which host plant the time was spent. In these two-choice tests the assays ran for 25 minutes. At the conclusion of both types of behavioural assays, the eight P. charybdis larvae were reared in a group, and the non-target larvae were also reared in a group on their host plant as described above. We aimed to conduct a minimum of 12 replicates on each species with the exception of T. sloanei, where insufficient larvae were obtained during the periods when E. daenerys was active, so only no-choice physiological and behavioural assays were conducted. The only assays discarded were those in which a female never contacted or attacked a single P. charybdis. After each assay in which larvae were attacked, they were also reared and fates recorded as for the physiological assays.

Recorded behaviours Time of each behaviour was recorded to the nearest second, as well as the time E. daenerys moved onto or off foliage, whether by flying or walking. From this we analysed the total time each parasitoid spent on each plant species foliage. Once a larva was encountered by the wasp the following behaviours were recorded: “attack-insertion” = a probe that involved the wasp curling its abdomen forward under its body, and inserting it into the larva for approximately one second; this is sufficient time for an egg to be laid (Rice 2005b). “attack-fail” = ovipositor probing behaviour that did not succeed in contacting the larva or maintaining contact adequately for successful egg deposition. “attack object” = ovipositor probing at beetle frass or less often at an exuviae or a piece of host plant such as a cut stem end. “disregard” = walked right over a larva without any apparent change in behaviour or else moved away from the larva after antennating it.

No female parasitoid was tested more than once against any one non-target species in the same assay type, but may have repeatedly been used in assays on subsequent days against other non-targets, and to oviposit into P. charybdis larvae to create positive controls.

Statistics To determine whether the number of attack-insertions or other count behaviours on the plant differed between beetle species, the Wilcoxon signed rank test was applied. This non-parametric hypothesis test was used in favour of the t-test because sample distributions could not be assumed to be normally distributed. The null hypothesis was that the median difference between pairs of behavioural observations was zero (i.e. equal medians). A 95% confidence interval was applied. The Wilcoxon statistic, W, and p- values are reported. The statistic W, in the comparison of “A” versus “B”, is the numerator in the estimated probability of the number of pairs of “A” being less than “B”. The denominator is simply the product of the two sample sizes. Because measures of central tendency such as the median, for which rates of attacks were identically zero for all non-targets, (Figure 3) or mean (more subject to outliers further from zero, but still low for all non-targets) fail (by definition of measures of central tendency) to identify behavioural extremes, we used alternative approaches (such as box and whisker plots) to visually express the behaviour shown. We have avoided potentially misleading stacked bar plots that would have been based on sample means. This approach allowed us to identify behavioural extremes in activity in addition to behavioural ‘norms’. Cases where boxes show a high degree of separation (i.e. no overlap), demonstrate significant differences in sample medians. Attack rate when only on the plant was also calculated as: Attack rate on plant = No. attack-insertions / total spent on that host’s plant. All analyses and data visualisation were performed using R (R Core Team 2017) base graphics, and the lattice library (Sarkar 2008) for data visualisation and display.

RESULTS Physiological Assays – 24 hours no-choice exposure The target pest beetle Paropsis charybdis negative controls (no wasp encounters) had a mean survival to pupation of 79%, with the survival rate varying between years due to a disease outbreak in 2016 (Table 2). The survival to pupation of P. charybdis larvae subject to a single attack-insertion event was less than 10%. Successful E. daenerys larval emergence, reared from P. charybdis, was between 30 - 34% (Table 2).

Table 2. Outcomes of 24-hour no-choice exposure of E. daenerys to non-target beetles reared to pupation, and comparison to negative controls of P. charybdis. Species No. hosts tested % larvae % beetles n dead larvae % died survival to contained parasitised unknown pupate Eadya by Eadya cause Pos control P. 32 56.2% 9.4% 0 34.4% charybdis 15 Pos control P. 208 62.8% 7.0% 0 30.2% charybdis 16 Trachymela sloanei 40 30.0% 57.5% 0 12.5% D. semipunctata 128 36% 62.5% 2 1.6% Allocharis nr tarsalis 80 2.5% 90.0% 6* 7.5% Chrysolina abchasica 112 58% 40.2% 2 1.8% Gonioctena olivacea 96 9.4% 85.4% 5 5.2% Lochmaea suturalis 88 26% 75.0% 0 0 Agasicles hygrophila 112 45% 55.4% 0 0 Neolema ogloblini 120 15% 85.0% 0 0 Cassida rubiginosa 128 40% 60.2% 0 0 Neg control P. 64 5% 95.3% 0 0 charybdis 15 Neg control P. 96 32% 67.7% 0 0 charybdis 16 *these 6 larvae had been killed for dissection, 2 others had died naturally and did not contain parasitoids

Overall, about two-thirds (5.4 out of 8) of all the non-target beetle larvae in each replicate successfully pupated (Table 2). The best rearing survival was achieved for A. nr tarsalis (90%), and the worst for C. abchasica (40%) (a problem also shared by Landcare Research, H. Gourlay, pers. comm.). Eadya daenerys only completed development, and emerged as a fully developed parasitoid larvae, from one non-target species, the Eucalyptus pest Trachymela sloanei. Three of the five emergent parasitoid larvae from T. sloanei spun tiny unviable cocoons (less than 5 mg), and a fourth spun a 12 mg cocoon that was viable and produced a very small adult wasp (head capsule width 1.04 mm wide) confirming T. sloanei as a physiological host. This can be compared to E. daenerys development through the target P. charybdis, where viable spun cocoons weighed a mean of 84.8 mg (range 46-137 mg, n=30) and the resultant adult head capsule width had a mean value of 1.48 mm wide (n= 61). Internal parasitism by E. daenerys was found in dead larva and prepupa of four non-target species, all from the subfamily Chrysomelinae: D. semipunctata, A. nr tarsalis, C. abchasica and G. olivacea (Table 3). Only A. nr tarsalis and C. abchasica contained any well-developed E. daenerys larvae, i.e. over 5 mm long. D. semipunctata and G. olivacea contained either first instar E. daenerys larvae or just the heads remained of the first instar parasitoids (Table 3). In A. nr tarsalis all instances of parasitism (n=6) were found in the prepupae that had failed to pupate, with E. daenerys larvae ranging from just first instar head capsules, to encapsulated first instars, through to two partially-developed larvae were about half the length of their host larva at 42 days after oviposition (Table 3). The decision to dissect the A. nr tarsalis prepupae was made after double the larval development time of E. daenerys had passed and the prepupae had long since ceased feeding. In parasitized C. abchasica larvae, death had occurred between 17 and 28 days in the larval stage with E. daenerys larvae being either small or medium sized.

Table 3. Immature E. daenerys recorded in dead larvae and dead prepupae of non- target beetles following 24-hour no-choice exposure to E. daenerys. Non-target % (n) larvae Range of days No. L1 E. No. >4mm species with immature until host daenerys per E. daenerys E. daenerys death larva per larva Dicranosterna 1.6% (2) 14-18 1,1 - semipunctata Allocharis nr 7.5% (6) Killed at 40-42 2, 5, 7, 17 1, 1 tarsalis Chrysolina 1.8% (2) 17 - 28 2 1 abchasica Gonioctena 5.2% (5) 18 - 45 1, 2, 3, 4, 6 - olivacea

Behavioural observations The order of presentation (A-B vs B-A) had no significant effect on the median number of attack-insertions, or the attack rate on the plant. Beetle species was far more important in determining the end result. In all but one case the number of larval attack- insertions was significantly higher towards target P. charybdis larvae than towards any non-target larvae (Figure 1). The only non-target species in which there was no significant difference between it and P. charybdis in the median number of attack-insertions in no-choice tests was T. sloanei (p=0.28, Wilcoxon test, median T. sloanei=1.5, median P. charybdis=2.5). The box-and-whisker plots (Figure 1) show a clear overlap of boxes only for P. charybdis compared with T. sloanei. All other paired non-target species received an identical median of zero attack-insertions, compared to the sometimes very high oviposition activity directed towards P. charybdis larvae, i.e. 30 attack-insertions in 10 mins in one no-choice test (Figure 1). The number of attack-insertions on P. charybdis larvae was greater during two- choice tests (a median of 8) than during no-choice tests (a median of 4) but they were not comparable assays due to different test durations. The E. daenerys attack rate on plant against P. charybdis larvae when on Eu. nitens foliage (means of between 2 – 5 attack-insertions per min) was higher than the attack rate against non-target larvae when on their host plant (mean of 0 – 0.8 attack- insertions per min), for the majority of species (Figure 2). The observations during assays suggested that the majority of the time E. daenerys was in contact with non-target plants bearing non-target larvae the parasitoid just sat and rested, or undertook grooming behaviour. The only exceptions where a difference did not occur were the attack rates on plant towards T. sloanei on Eu. nitens foliage compared to P. charybdis, and towards A. nr tarsalis while on V. albicans foliage in two-choice tests (Figure 2). In the latter case two female parasitoids were responsible for this attack behaviour, and in both cases these individuals undertook more attack-insertions against P. charybdis larvae than they did against A. nr tarsalis larvae. The median most common attack rate on plant was substantially different, but some outliers created an overlap in the upper quartiles. In the presence of T. sloanei, the majority of female E. daenerys (5 out of 8, or 62.5%) were actively involved in attack-insertion on T. sloanei larvae at least once. In all other non-target species in the no-choice assays, an average of only 8.4% (range 0 – 25.0%) of female E. daenerys tested attack-insertion on a larva at least once. In two- choice assays (which did not include T. sloanei) the average was 14.7% (range 0-35%) of E. daenerys females conducted attack-insertion on non-target host compared to 100% undertaking attack-insertion on P. charybdis.

Figure 1. Box and whisker plots representing total number of successful attack- insertions counted for each E. daenerys in no-choice sequential and two-choice tests. The P. charybdis target larvae are shown in grey, while the non-target species they were paired against are coloured (red to orange colours depict the subfamily Chrysomelinae, greens subfamily Galerucinae, and blues other subfamilies). The number of replicates (N) tested (with eight beetle larvae per replicate), the Wilcoxon test statistic (W) and significance levels for each pair are given on the right hand side of each row. The vertical line in each box represents the median, and the box represents the midspread, with the whiskers extending to the minimum and maximum values. Where no box is visible, just a line, it means that all the data points are sitting at, or close to, zero. The mean number of attack-insertions is represented by an asterisk.

Figure 2. Box and whisker plots of Eadya daenerys ‘attack rate on plant’ behaviour directed against a beetle species in no-choice and two-choice tests. The P. charybdis target larvae are shown in grey, while the non-target species they were paired against are coloured (red to orange colours depict the subfamily Chrysomelinae, greens subfamily Galerucinae, and blues other subfamilies). The number of replicates (N) tested (with eight beetle larvae per replicate), the Wilcoxon test statistic (W) and significance levels for each pair are given on the right-hand side of each row. The vertical line in each box represents the median. The box represents the midspread, with whiskers extending to the minimum and maximum values. Where no box is visible, just a line, it means that all the data points are sitting at, or close to, zero. The mean attack rate on plant is represented by an asterisk.

We also examined the average number of behaviours counted as undertaken by E. daenerys females per species and per test type. The shaded bars reveal the range of average scores obtained per species test. As with the non-parametric statistics, averages follow the same trends with little or no overlap between P. charybdis and non-target species (Figure 3). Again the only exception to this is the number of attack-insertions and number of attack-fails directed towards T. sloanei in the no-choice assays. The proportion of female E. daenerys exhibiting attack-insertions, attack-fails and object attack behaviours was relatively high. For example, 3 out of 8 E. daenerys did object attacks and 4 out of 8 did attack-fails when in the presence of T. sloanei. The only behaviour E. daenerys exhibited that was counted more in the reps with non-target species compared with P. charybdis was the “disregard” of larvae encountered (Figure 3). For example, half the female E. daenerys disregarded D. semipunctata larvae that they encountered on blackwood foliage in no-choice tests and 12 out of 17 females disregarded D. semipunctata larvae in choice tests, compared to only 5 of the same females that disregarded one or more P. charybdis larvae during the paired two-choice test. One exception to the above trend was the behaviour E. daenerys exhibited with regard to object attacks when in the presence of C. abchasica in no-choice tests. In fact 12 of the 15 individual E. daenerys participated in object attacks (during the assay C. abchasica larvae laid large round black frass pellets of a similar size to a paropsine larva, which may have been a visually stimulating cue for E. daenerys females that stimulated them to attack. Some object attacks were also directed towards the cut end of a tutsan twig).

Figure 3. Overall behavioural differences in Eaedya daenerys during observed no- choice and choice tests. Each box shows the range of counts of each behaviour per test, with the shaded area showing the range of average behaviours in that activity across the paired target/non-target species.

Rearing of target and non-target replicates when attack-insertions were observed against those larvae during the above behavioural observations adds a little more data to the physiological host range development data from the 24 hour tests (Table 3). Many P. charybdis larvae died after these assays due to multi- parasitism (being over-stung) as the number of attack-insertions at times exceeded 30 (Figure 3). Observed attack-insertions by E. daenerys against C. abchasica resulted in three dying larvae being internally parasitized, from which no E. daenerys emerged. Whereas another five attacked larvae showed no evidence of parasitism. Again one of the E. daenerys larvae was large and the remainder were small. The same pattern as observed previously was also repeated with A. nr tarsalis larvae; observed attack-insertions in behavioural assays resulted in four of the larvae reaching the pre-pupal stage but failing to pupate. These had been killed for dissection after double the period of time needed for parasitoid development had passed. Five of the dissected larvae contained very small or encapsulated E. daenerys larvae in four cases, and one contained a moderate, well-developed E. daenerys (Table 4). Another three attacked A. nr tarsalis larvae were reared and showed no evidence of parasitism (one died, two became adult beetles). No other non-target larvae, even those observed to have been the subject of an attack-insertion in observed assays, were found to have been parasitized.

Table 4. Outcomes of additional rearing from no-choice (NC) and two-choice tests (2) following observed E. daenerys attack-insertions on beetle larvae. Species Test No. Dead % beetles % (n) dead % Eadya larvae larvae survival to larvae emerged reared unknown pupate contained causes Eadya Paropsis 2016 NC 369 34.0% 43.8% 0.8% (3) 21.4% Paropsis 2017 NC 381 29.1% 47.7% 2.1% (8) 21.1% Paropsis 2018 NC 104 17.3% 65.4% 0 17.3% Paropsis 2016 2 344 18.9% 34.6% 1.1% (4) 28.2% Paropsis 2017 2 504 31.5% 39.5% 1.8% (9) 27.2% Paropsis 2018 2 56 10.7% 66.1% 0 23.2% T. sloanei NC 12 41.7% 58.3% 0 0 D. semipunctata NC 8 25% 75% 0 0 D. semipunctata 2 27 40.7% 59.3% 0 0 C. abchasica NC 9 11.1% 66.7% 22.2% (2) 0 C. abchasica 2 11 63.6% 27.3% 9.1% (1) 0 A.nr tarsalis NC 3 0 33.3% 66.6% (2) 0 A.nr tarsalis 2 16 6.3%* 75% 18.7% (3) 0 Gonioctena NC,2 0 - - - - Lochmaea NC 26 53.8% 46.2% 0 0 Agasicles NC 3 33.3% 66.7% 0 0 Agasicles 2 16 37.5% 62.5% 0 0 Neolema NC 8 12.5% 87.5% 0 0 Neolema 2 33 24.2% 75.8% 0 0 Cassida 2 8 50% 50% 0 0 *one of these larvae died naturally but did not contain parasitoids

All negative control larvae that were reared during the experimental processes were weighed when they reached the prepupal and pupal stages. The data show that P. charybdis pupae weighed one and a half times (mean of 153 mg) the next largest species, D. semipunctata (91.2 mg), followed by the small tortoise beetle T. sloanei (mean of 38.4 mg) (Figure 4). The rest of the non-target species pupal weights were significantly less.

Figure 4. Comparison of pupal weight of each uninfested target or non-target beetle species reared in the laboratory. The vertical line within each box represents the median. The box represents the midspread, with whiskers extending to the minimum and maximum values. The mean pupal weight for each species is represented by an asterisk.

DISCUSSION Physiological host range

Physiological host range testing was undertaken in such a way as to maximise the chances of Eadya daenerys attacking non-target larvae, informing the worst case scenario risk. This is accepted as best practise for biological control of arthropods (van Lenteren et al. 2006). It would have been inappropriate to undertake an identical procedure for the positive controls with Paropsis charybdis larvae, as 24 hours enclosed with an E. daenerys would be too long with just eight larvae, and would have resulted in multi- parasitism and premature death. The positive control data is from P. charybdis second instar larvae having been stung just once by one parasitoid before being reared to pupation or emergence of an E. daenerys parasitoid larva. Using this method, P. charybdis was shown to be an excellent physiological host and E. daenerys was capable of reducing beetle survival to pupation from approximately 80% when unparasitised, down to approximately 8% when parasitised. The small tortoise beetle T. sloanei was the only other complete physiological host for E. daenerys as revealed from the host range testing. This pest is nocturnal (Tribe & Cillié 1997) and larvae hide together in clusters during the day in either bark crevices or leaf rolls, thus our assays made them artificially available for attack during the day. The T. sloanei larvae also did not readily settle. During the no-choice observations their continual movement made them a difficult target for E. daenerys to complete attack- insert behaviour. While only one E. daenerys larva completed development to pupation in T. sloanei, five had matured to emergence from the beetle larva. Many parasitoid species attack hosts sharing an ecological niche (Memmott et al. 1994) and certainly this species shares a very similar niche to P. charybdis, but because the larvae only feed during the night it is uncertain to what extent E. daenerys would be able to use T. sloanei, or whether E. daenerys females using/emerging from T. sloanei have equal fitness to those using P. charybdis as a host. Whether or not this is a false positive result that is purely a laboratory artefact may not be known until the E. daenerys host range can be further assessed in the field. Trachymela spp. have never been reported as hosts of any Eadya spp. in Tasmania, though in our more recent field work, none were collected or reared (Peixoto et al. 2018). Given that T. sloanei is an exotic pest of Eucalyptus in New Zealand, it being a possible physiological host for E. daenerys does not detract from the value of the proposed introduction, but rather increases its potential usefulness.

Geographic and niche overlap Phylogeny is not the only factor that influences the parasitoid host range (Haye et al. 2005) but it is currently the best predictor with which to initiate assessments of the risk posed to non-targets. Although most of the non-target species tested against E. daenerys were exposed leaf-feeding larvae on weeds and shrubs that could occur in the same environment as Eucalyptus trees, only T. sloanei inhabits a similar niche to P. charybdis, and uses the same host plants. All the non-target species tested (Table 1) do have the potential to geographically overlap with P. charybdis in some habitats in New Zealand. All the other host plants especially the weeds are, by definition, common in the environment. Phylogeny did play an important role as all the non-target species that revealed some internal parasitism by E. daenerys, even though they did not support complete development of the parasitoid, were in the Chrysomelinae: D. semipunctata, C. abchasica, G. olivacea and A. nr tarsalis. Dicranosterna semipunctata, the blackwood tortoise beetle, is an exotic pest of A. melanoxylon, and acacias are often grown in similar habitats to eucalypts in New Zealand, such as in farm forestry plantations, creating geographical overlap with eucalypts. In the case of C. abchasica, its host plant is the weed Hypericum androsaemum (tutsan), which is also present in the North Island in farming and forestry areas, so is likely. The tutsan leaf beetle, however, is yet to be confirmed as established in the field in New Zealand, following release in 2017 (H. Gourlay pers comm). The broom leaf beetle, G. olivacea, also has high potential for geographical (but not niche) overlap with eucalypts in New Zealand. Although the biological control agent is currently not widely established (H. Gourlay pers comm), the weed host Scotch broom is widespread and extremely common, suggesting that the range of G. olivacea may yet expand. In contrast to the above, it is extremely unlikely that the endemic beetle A. nr tarsalis will ever overlap with eucalypts, due to its restricted distribution in the subalpine zone (1,200–1,400 metres) in Kahurangi National Park, New Zealand (Wardhaugh et al. 2018). To the best of our knowledge no sub-alpine Eucalyptus species such as snow- gums being grown in New Zealand for plantations, and there are no wilding eucalypts in Kahurangi National Park. The closest eucalypts are likely to be present in the Motueka River Valley, directly east of the park and approximately 6 km away, but 1100m lower in elevation. We surmise therefore that the only potential for E. daenerys to geographically overlap with A. nr tarsalis would be if individual parasitoids were to be blown via an easterly updraft into the sub-alpine zone where A. nr tarsalis and its host plant Veronica albicans grow. Since no hosts are present in the vicinity, E. daenerys will not be able to form a self-sustaining population in those areas inhabited only by A. nr tarsalis. Indeed, the latter has proven not to be a physiological host for this parasitoid. With E. daenerys unable to establish, the potential for any negative impacts on A. nr tarsalis is limited to mortality of individual larvae that get stung and fail to complete their development. This scenario is likely to be an extremely rare event, especially in light of the low propensity of females to attack A. nr tarsalis (Figures 1 and 2).

Body size comparison Many factors influence the physiological host range of E. daenerys, starting of course with whether females are stimulated to oviposit into any non-target species. Once an egg is deposited into the haemocoel of the host, internal chemistry and host resistance determine whether the larva will be viable. In the absence of host resistance, the size that the host larva will attain, and the duration of host larval development may become limiting factors. Paropsis charybdis is the largest known host of E. daenerys (Table 1) (Peixoto et al 2018), however E. daenerys primarily utilises early-instar Pst. agricola (Rice 2005b) in Tasmania. Research has revealed that the parasitoid has almost identical thermal requirements to this host, and a developmental rate that is slightly faster (Rice & Allen 2009), meaning it avoids the constraint of outgrowing its host larva. The prepupal weight of Pst. agricola ranges from 21 - 89 mg (mean 55.9 mg) (Smart 2017). Furthermore the weight of the host prepupae will influence the size of the emerging E. daenerys (Smart 2017). Paropsis charybdis at mean 154 mg pupal weight (Table 1), is significantly larger (Figure 4) than all the other non-targets tested. However, the minimum size to accommodate complete physiological development of E. daenerys, is estimated at approximately 35 mg, which encompasses both Pst. agricola (Smart 2017) and T. sloanei (Figure 4). All the other species of non-target beetles tested (Table 1) apart from D. semipunctata, are significantly smaller than this, none exceeding 31 mg maximum pupal weight (Figure 4). This adds to our confidence that no non-target beetle present in New Zealand that matures to less than 35 mg pupal weight will be able to support complete development of E. daenerys.

Larval development rate The rate at which non-target species develop as larvae may also impact on their ability to be hosts. Eadya daenerys develops in 25 days within its host at 18˚C (Rice & Allen 2009), but this speeds up at higher temperatures. At a constant 22˚C, development to emergence occurs in 18 days (Rice & Allen 2009). We do not have the same detailed data on rates of development in response to temperature for all the non-target beetles tested, but any non-target larvae that develop more rapidly than E. daenerys at the same temperature (approx. 18-25 days), or whose pupal body weight is less than the estimated minimum of 35 mg, are extremely unlikely to support physiological development of the larval stage of E. daenerys (Table 1).

Behavioural attraction to non-targets Host plant odour volatiles from Eucalyptus likely play a key role in host location by E. daenerys, as odour cues are known to be critical in many parasitoid host-finding behaviours (Turlings et al. 1993; Avila et al. 2015). In the no-choice assays E. daenerys directed the same number of attack-insertions towards the two Eucalyptus-feeding species P. charybdis and T. sloanei. Whereas Dicranosterna semipunctata incurred attacks from only one third of E. daenerys females. The proportion of E. daenerys attacking the other Chrysomelinae (A. nr tarsalis or C. abchasica) was closer to approximately one in five parasitoids. Apart from T. sloanei, all other non-target species were significantly less likely to receive an attack-insertion, an attack-fail, or for their frass or exuviae to be probed (object attack) compared to P. charybdis (Figure 3). This was consistent across all assay methods trialled, both no-choice and two-choice. This suggests that without the odour cues associated with being a beetle species that feeds on Eucalyptus foliage, other chrysomeline larvae will not attract the attention of E. daenerys. Eadya daenerys used in our tests were of unknown mated status and unknown age which may have influenced parasitoid behaviour. A large degree of variability in activity was observed amongst E. daenerys in all tests and we are not overly concerned by the occasional individual undertaking attack-insertions on non-targets when the majority did not. It is likely the less discriminating individuals were highly deprived (Barton Browne & Withers 2002). For instance, the four female parasitoids that undertook attack- insertions on Neolema ogloblini almost all did so after more than two weeks of being held in captivity. By having sufficient replication, we were able to interpret these results as potentially laboratory artefacts, and looking at the whole data set they fall into place as not statistically significant.

Risk assessment In assessing possible risk to non-target species from E. daenerys, the spatial and temporal characteristics of the target and agent need to be considered (Kuhlmann et al. 2006). The host Paropsis charybdis occurs throughout New Zealand but it only feeds on Eucalyptus, which is an exotic tree genus in New Zealand, and is generally not present in native forests. However, as discussed above, it may geographically overlap with some of the non-target weed biocontrol agents we have tested. Research indicates that colonization by parasitoids (parasitism) of weed biological control agents can best be predicted as being those agents with “ecological analogues” in the field in New Zealand (Paynter et al. 2010). Paynter et al (2010) defined an ecological analogue as a New Zealand insect that (i) is taxonomically “closely” related to the agent; (ii) has a similar lifestyle niche; and (iii) feeds on the target weed. Paynter et al. (2010) refer to analogues as native insect herbivores feeding on the plant of interest, but we propose it is just as relevant to consider exotic pest insect herbivores too, especially those that have parasitoids established, or in our case, about to be established. Interpolating from the angle of what risk our introduced parasitoid could present to weed biological control agents in New Zealand, we need to consider the question: Is our target pest P. charybdis an ecological analogue of any leaf-feeding chrysomelids? To be an ecological analogue this beetle would need to use a woody Myrtaceae, preferably a Eucalyptus, from time to time. None of the beneficial weed biological control agents we considered attack Myrtaceae host plants (Table 1) and all are significantly smaller (Figure 4). Furthermore, they feed on unrelated shrubs, rather than trees. Using this criterion, there are no weed biological control agents in New Zealand likely to be at risk of E. daenerys parasitising them in the future. Furthermore the host testing results (Table 2) revealed that none of the weed biological control agents that were attacked under laboratory conditions were complete physiological hosts. This indicates that no self- sustaining populations of E. daenerys will parasitise any weed biological control agents in New Zealand, and would only become a risk in the future should a closely-related Myrtaceae tree become a target for a weed biological control programme. This appears highly unlikely. We conclude that E. daenerys carries a low risk to non-target species and thus is safe to release into New Zealand.

ACKNOWLEDGEMENTS Many thanks to other Scion staff including Andrew Dunningham, Justin Nairn, Matt Scott, Pam Taylor, Carl Wardhaugh, and UTAS staff members Vin Patel and Steve Quarrell. Dean Satchell has also been an invaluable part of this research. Students have also been extremely valuable to this project, in particular Mike Davy, Sean Gatenby, Katherine Moors, Elise Peters and Amy Yasutake-Watson, in New Zealand, and Hui Law, Meng Lim, Allana Russell, and Rebekah Smart, in Australia. Thanks to Landcare Research scientists Ronny Groenteman, Hugh Gourlay, Chris Winks, Paul Peterson and Richard Leschen and AgResearch scientist Mike Cripps for their invaluable assistance with non-target beetles. Allocharis nr tarsalis were collected under DOC research authority 54216RES. Funding was from the NZ MPI Sustainable Farming Fund contracts 12-039 and 407964, and NZ Ministry of Business Innovation and Employment Strategic Science Investment Funding contract to Scion. Industry funding co-partners Southwood Exports Limited and Oji Fibre Solutions NZ Ltd (previously Carter Holt Harvey fibre solutions) are gratefully acknowledged, as are the Speciality Wood Products Partnership, and the NZ Farm Forestry Association.

Supplementary data 1: Summaries of the life history of each beetle tested

Paropsis charybdis Stål Chrysomelinae: Chrysomelini Paropsini are primarily larval and adult herbivores of eucalyptus. This target species specialises in feeding on the flush foliage of Eucalyptus: Symphyomyrtus species, which have economic importance as exotic forestry species in New Zealand. Paropsis charybdis undergoes two generations per annum, with larval peaks occurring from November to December, and February to March (Bain & Kay 1989; Murphy & Kay 2000; Jones & Withers 2003). Adult generations overlap, as evidenced from the multi-generational infections of phoretic mites in their native range (Seeman & Nahrung 2013). Paropsis charybdis larvae can be found from early spring until late autumn. Eggs are laid in rows at the tip of mature adult foliage, from where hatching neonates eat their egg casings, then disperse to reach the newest flush of adult foliage at the top of the tree. Each instar can consume progressively more expanded/mature leaves (McGregor 1984). All four larval instars are solitary feeders, and exhibit strong defensive behaviour by everting dorsal glands between the eighth and ninth dorsal segments when disturbed (Clark 1930). When larvae complete feeding they drop to the ground and pupate within the leaf litter (McGregor 1984).

Trachymela sloanei (Blackburn) Chrysomelinae Chrysomelini Trachymela sloanei has also been a defoliating pest of Eucalyptus trees since invading New Zealand in 1976. Both the larval and adult stages of the beetle feed on a wide variety of eucalyptus (Selman 1985), with preferred hosts in California, where it is also an invasive pest, listed as Eucalyptus globulus and E. viminalis (Millar et al. 1999). The species has since spread from California into Spain and Mexico. The eggs of T. sloanei are small and red-brown and laid in a cluster stuck together in bark crevices. Hatching larvae are initially gregarious and feed nocturnally on young leaves of their host plants. They feed through four instars; later instar larvae become solitary but remain nocturnally active (Selman 1985). Trachymela tincticollis larval feeding has been studied in detail (Tribe & Cillié 1997) revealing they leave their shelters to feed twice per night, once at dusk, and one just before dawn. It is likely T. sloanei feeds in the same manner (T. Withers, pers. obs). Pupation occurs within leaf litter and adults overwinter beneath bark. It is likely they are bivoltine, with overlapping generations, like the closely related Trachymela catenata (Chapius) (Barrett 1998). Trachymela sloanei prefers many of the same eucalypt species as the target, and multivoltinism suggests niche and habitat overlap with the target and therefore the proposed biocontrol agent. However there is no evidence that E. daenerys is nocturnally active, and therefore encounters with T. sloanei larvae may be rare.

Dicranosterna semipunctata (Chapuis) Chrysomelinae: Chrysomelini Dicranosterna semipunctata is a pest of the exotic tree species Acacia melanoxylon R. Br. (Fabales: Mimosidae). Native to Australia (Simmul & De Little 1999), it invaded New Zealand in 1996 (Murray & Withers 2011). It has also been recorded feeding occasionally on Acacia koa A. Gray, Acacia implexa and Paraserianthes lophantha (Willd.) I.C. Nielsen. Dicranosterna semipunctata is probably univoltine in New Zealand, within oviposition beginning in spring (October to December) with the onset of new A. melanoxylon phyllode growth. Eggs are laid singly on the foliage, and larvae proceed through four larval instars, feeding solitarily. Pupation occurs in the leaf litter and adult beetles reappear from diapause in late summer and feed heavily on foliage (Murray & Withers 2011). A second generation in late summer has not been proven. The phenology of this spring generation and feeding behaviour suggests high potential for geographic overlap with the proposed biocontrol agent, as eucalypts are often planted within a similar habitat to A. melanoxylon across rural New Zealand.

Allocharis nr tarsalis Chrysomelinae: Chrysomelini This species is a naturally uncommon native beetle Allocharis nr tarsalis (Det. Richard Leschen, Landcare Research, pers. comm. 2018). Scion scientists collected larvae and adults that were feeding on the foliage of Veronica (Hebe) albicans Cockayne (Lamiales: Plantaginaceae), which grows in the mountainous region of Kahurangi Nation Park, near Nelson, New Zealand, in the subalpine zone at 1,200–1,400 metres. The beetle is thought to be univoltine with overwintered adults emerging from hibernation in leaf litter in early summer. After hatching from eggs, the black larvae feed diurnally on young foliage during December and January, before pupating in the leaf litter and probably entering extended diapause (Wardhaugh et al. 2018). Adult beetles from this same genus have previously been collected at night by beating sub-alpine foliage (NZAC specimen labels) in the South Island, and larvae of one other similar species Allocharis robusta Broun were collected off “flowers and leaves of mountain Veronica’s” at Mount Cook village in 1928 (Hudson 1934).

Chrysolina abchasica (Weise) Chrysomelinae: Chrysomelini The larvae and adults of the beetle Chrysolina abchasica feed on the leaves of tutsan, causing defoliation. Tutsan (Hypericum androsaemum L., Malpighiales: Hypericaceae) is an evergreen or semi-evergreen shrub that grows to about 1.5 m tall (Groenteman 2013). Tutsan was first recorded as naturalised in New Zealand in 1892. It was noted as a weed of local significance in 1955, but since then has become a serious weed in the North Island of New Zealand. It has taken much sheep-grazed hill country out of production, especially in the Ruapehu district. Chrysolina abchasica was approved for release as a beneficial weed biological control agent in New Zealand in 2016 (Hayes 2007). Chrysolina abchasica is a univoltine, leaf-feeding beetle introduced from Georgia (Europe)(Bieńkowski 2011). Eggs hatch immediately upon laying within the leaf, and the first instar immediately eats out of its oviposition site. The remaining instars feed solitarily until the fourth instar drops off to pupate in the soil (Hayes 2007). Further field releases in New Zealand are planned for 2018/19 (H. Gourlay, pers. comm).

Gonioctena olivacea (Förster) Chrysomelinae: Chrysomelini In 2007 Gonioctena olivacea was introduced from the UK as one of the biological control agents for controlling the European legume scotch broom Cytisus scoparius (L.) Link (Fabaceae). Broom has been in New Zealand since naturalising in 1872, is unpalatable to livestock, and dense infestations have negatively affected productive and conservation lands (Syrett et al. 1999). Beetles are univoltine, adults appearing in spring to early summer and laying eggs on the leaf surface of host plants. Larvae feed on leaves and attain full growth through three larval instars. The fully grown larvae drop off the plant to pupate in the soil. When new adults emerge in summer they maturation- feed before entering winter diapause (Biedermann 2005). Establishment is currently limited to a few sites in north the north and south islands of New Zealand (Hayes 2007).

Lochmaea suturalis (Thomson) Galerucinae: Luperini Lochmaea suturalis was introduced in 1996 from Oakworth, northern UK, for the biological control of heather, Calluna vulgaris (L.) Hull (Ericaceae). Heather, having been introduced to New Zealand, was rapidly spreading to significantly impact upon the natural sub-alpine ecosystems of Tongariro National Park (Syrett et al. 2000). The beetle is univoltine, adults becoming active when temperatures exceed 9˚C. Eggs are laid within the damp moss layer at the base of plants. Larvae feed rapidly through four instars on the tiny leaves on heather shoots, before pupating within the leaf litter. Newly emerged adults also feed on leaves, before entering the leaf litter to undergo winter diapause, emerging again in approximately November. The heather beetle has performed relatively poorly in New Zealand, which may be a result of the beetles having gone through a genetic bottleneck during their introduction (Fowler et al. 2015).

Agasicles hygrophila Selman & Vogt Galerucinae: Alticini Agasicles hygrophila (1981) was introduced in 1984 from Argentina as a beneficial insect against the problem semi-aquatic rooted weed of northern areas of New Zealand, Alternanthera philoxeroides (Mart.) Griseb. (Caryophyllales: Amaranthaceae). Both adults and juveniles feed on the leaves of A. philoxeroides and occasionally Alternanthera denticulata R. Br. A (Paynter et al. 2017), defoliating the plants. Although the weed is aquatic, it can also spread to infest damp soil beyond the edges of infested water bodies. Larvae feed for a short period of just two weeks through three larval instars before boring a hole in the stem and entering the stem to pupate. The entire life cycle can be completed in less than one month. They have been known to achieve substantial control of alligator weed in still bodies of water such as shallow lakes (Philip et al. 1988). They are restricted to sites in the upper north island.

Neolema ogloblini (Monrós) Criocerinae Neolema ogloblini was introduced into New Zealand from Brazil in 2011 as a biological control agent for the weed Tradescantia fluminensis Velloso (Fowler et al. 2013), commonly known as tradescantia. Tradescantia is a major warm temperate understory environmental weed, forming mats that prevent forest regeneration. Adult beetles are 4- 5mm long and survive for a few months. Eggs are laid in small clusters on the underside of leaves, hatching into pale brown larvae. The larvae accumulate a faecal shield on their dorsal surface. Larvae of the first few instars sometimes feed gregariously on the leaf surface, but separate when they are older, feeding individually on the leaf. The final larval instar pupates within a pupal cocoon attached to the leaf, resembling white styrofoam. Development from egg to adult can occur within 6 weeks under warm temperatures. It is believed to undergo 2-3 generations per annum (Hayes 2007).

Cassida rubiginosa Müller Cassidinae:Cassidini Cassida rubiginosa was introduced into New Zealand from Europe in 2008 as a beneficial insect against thistle pasture weeds (mainly Cirsium arvense (L.) Scop. (Asterales: Asteraceae). The beetle is univoltine with overwintered adults emerging from hibernation in early spring. Females lay oothecae, containing about ten eggs, on the undersurfaces of thistle leaves during spring. Larvae are leaf feeders, and pass through five instars, pupating attached to the leaf (Koji et al. 2012). Larvae accumulate a faecal shield on their dorsal surface, which has been shown to assist with disrupting oviposition from the hymenopterean parasitoid Forsterella reptans in Europe (Bacher & Luder 2005). REFERENCES

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