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