Selecting potential non-target species for host range testing of Eadya paropsidis

Transcription

1 Benefits and challenges of insect biocontrol 179 Selecting potential non-target species for host range testing of Eadya paropsidis T.M. Withers 1, G.R. Allen 2 and C.A.M. Reid 3 1 Scion, Private Bag 3020, Rotorua 3046, New Zealand, 2 School of Land & Food/TIA, University of Tasmania, Private Bag 54, Hobart TAS 7001, Australia 3 Entomology Dept., Australian Museum, 6 College Street, Sydney, NSW 2010, Australia Corresponding author: toni.withers@scionresearch.com Abstract Classical biological control is proposed for Paropsis charybdis (Coleoptera: Chrysomelidae: Chrysomelinae), a eucalypt pest established in New Zealand. The Australian solitary larval endoparasitoid Eadya paropsidis (Hymenoptera: Braconidae) is under investigation. A potential non-target species list was compiled for host range testing. There are no endemic species of paropsines in the New Zealand fauna, only invasive pest beetles. The most closely related endemic beetles to the paropsines are Chrysomelinae in the genera Allocharis, Aphilon, Caccomolpus, Chalcolampra and Cyrtonogetus. Little is known about these species. New Zealand has also introduced 12 beneficial chrysomelid weed biological control agents, which include Chrysomelinae and their sister group the Galerucinae. One endemic beetle, six beneficial beetles and two pest beetles are listed as the highest priority species for host specificity testing. Keywords biological control, host specificity testing, Chrysomelidae, paropsine. INTRODUCTION Host range testing prior to introducing a classical biological control agent provides an estimate of the risk of negative impacts on non-target species Barratt et al. (1999). Phylogeny is a valuable starting point for predicting and assessing the field host range of a parasitoid (Hoddle 2004), but other criteria such as ecological similarities are also very important (Kuhlmann et al. 2006). Kuhlmann et al. (2006) proposed developing an initial list of all potential non-target species based on phylogenetic affinities, ecological similarity to the target, and socioeconomic considerations. This list is then filtered by spatial, temporal and biological attributes such as size that might make a species effectively inaccessible to the proposed biological control agent, and secondly by the feasibility of obtaining laboratory colonies of the non-target species for testing. During host range testing, any new information gathered (such as attack by the proposed agent on one of the non-target species) may alter the type or extent of testing required, and potentially reduce or increase the final list of non-target species that are actually screened (Kuhlmann et al. 2006). This approach will be closely followed for obtaining a list of non-targets for host range testing of Eadya paropsidis Huddleston & Short (Hymenoptera: Braconidae). Paropsis charybdis Stål is a eucalypt defoliator from Australia that has been present in New New Zealand Plant Protection 68: (2015) New Zealand Plant Protection Society (Inc.) Refer to

2 Benefits and challenges of insect biocontrol 180 Zealand (NZ) since 1916 (Bain & Kay 1989) and continues to be the most significant pest of eucalypts throughout the country. In particular Eucalyptus nitens (Deane et Maiden) Maiden plantations from Southland to the central North Island can be heavily defoliated, and numerous other Eucalyptus species in warmer regions are also highly palatable to the pest. The cost of managing P. charybdis (Withers et al. 2013) is a risk for new forest plantations being developed for timber, pulp and paper. Egg parasitoids of P. charybdis have proven inadequate for population suppression (Mansfield et al. 2011). Tasmania was selected as the source area for a renewed search for natural enemies of P. charybdis, because this species outbreaks occasionally there (De Little 1989). Also Tasmania is known to be a good climatic match to plantation forest areas of NZ (Murphy 2006). Three years of field and laboratory research identified the most promising agent to target first generation larvae of P. charybdis as the parasitoid, E. paropsidis (Withers et al. 2012). Eadya paropsidis is a solitary larval koinobiont parasitoid specific to Paropsis and Paropsisterna species (Coleoptera: Chrysomelidae: Chrysomelinae) in Australia (Rice 2005a). It is a medium sized (ca 10 mm) black wasp with a bright orange head. Eadya paropsidis oviposits small (0.16 mm), hydropic eggs directly into the haemocoel of its hosts, and can attack all larval instars (Rice 2005b). Eggs of E. paropsidis hatch in around 5 days at 22 C (Rice 2005a). Developmental rates of E. paropsidis from egg insertion in the host to pupation in Paropsisterna agricola (Chapius) have been determined over a range of temperatures and average 21 days at 20 C (Rice & Allen 2009). Eadya paropsidis emerges from the host s prepupal stage, spins a silk cocoon, and then undergoes an obligate pupal diapause for around 10 months until the following summer (Rice 2005a). Information (Rice 2005b) collected from one field site over 2 years suggests E. paropsidis is univoltine in Tasmania, with adults present in November and December. Recent field collections reveal it is widely distributed from sea level (near Hobart) to 600 m (near Cradle Mountain) (G.R. Allen, unpublished data). The peak adult stage in December coincides with the peak early instar larval stage of many paropsines, including the abundant P. agricola (Rice 2005b). In 2014 Eadya paropsidis was imported into containment in NZ for rearing so that host range testing could begin. This paper outlines the process for developing a list of non-target species for host range testing. METHODS An analysis of the NZ coleopteran fauna and published phylogenies was conducted to establish which species of Chrysomelidae present in NZ had the closest taxonomic affinities to P. charybdis. To reduce all these potential non-target species down to a testing list, the filters of spatial, temporal and biological attributes were then applied. Spatially, those species present in the same or overlapping habitats to P. charybdis can be identified, but any non-target species cannot be ruled out at this stage because there is no information on the propensity for E. paropsidis to search for hosts in other habitats. Furthermore, P. charybdis has a nationwide distribution in NZ, wherever eucalypts are grown, which includes trees on the margin of other habitats. Temporally, species can be filtered down to those with larvae present when the adult parasitoid E. paropsidis will be active. The target pest P. charybdis is broadly bivoltine in NZ. Larvae are present from November to December, and February to March (Bain & Kay 1989; Murphy & Kay 2000; Jones & Withers 2003), with overlapping adult generations. Rice (2005b) documented adult E. paropsidis activity in the field from adult emergence traps, malaise traps and parasitised larvae collections and found adults to be active from early December to early February but peaking in December. Adult E. paropsidis collected in the field live for a maximum of 24 days in the laboratory (Rice 2005a). Based on climate matching the phenology of E. paropsidis is expected to be identical in both countries. In summary, only non-target species with larvae present in early to mid summer are under likelihood of attack. The final filter to be applied to predict risk to non-target species is biological attributes. Size is 2015 New Zealand Plant Protection Society (Inc.) Refer to

3 Benefits and challenges of insect biocontrol 181 the most obvious. Eadya paropsidis has been reared successfully in the laboratory on a wide range of host larval sizes, with ovipostion into hosts ranging in size from first instar P. agricola (ca 0.5 mg) through to final instar P. charybdis (ca 120 mg) (G.R. Allen, unpublished data). Eadya paropsidis prefers early instars of P. agricola to parasitise and reaches on average around mg pupal weight in this host (G.R. Allen unpublished data; Rice 2005b). Minimum viable host size limits have not yet been ascertained so non-target species cannot be ruled out at this stage based on larval size. The only biological attributes required for non-target larvae are that they have a leaf feeding mode (E. paropsidis searches on eucalypt leaves for host larvae), and are exposed during the day when E. paropsidis is active. Potential non-target species present in NZ are taken through these filters to produce a revised list of species for host range testing against E. paropsidis. RESULTS AND DISCUSSION Categories for consideration of non-target species The categories used for selecting an initial test list include ecological similarities, phylogenetic relatedness and safeguarding of beneficial insects (Kuhlmann et al. 2006). The ecological similarity category includes beetles with external leaf-feeding larvae that feed on Eucalyptus spp. in NZ, and includes several pest paropsine species: Trachymela catenata (Chapuis) and Trachymela sloanei (Blackburn) (Murray et al. 2010), and Paropsisterna beata (Newman) (Kean et al. 2015) (currently under an eradication campaign). Also present is the Australian gum tree weevil Gonipterus platensis Marelli (formerly G. scutellatus). It feeds on the same host species as P. charybdis and the exposed larvae are leaf feeders in late spring (Nuttall 1989), but being a curculionid it may be too distantly related to paropsines to warrant further consideration (Kuhlmann et al. 2006). No native beetles with leaf-feeding larvae feed on Eucalyptus or other Myrtaceae. Ecological similarity can also include insects associated with plant species found in directly adjacent habitats. Australian Acacia species are often planted mixed with Eucalyptus in NZ. Beetles that feed on Acacia in NZ include Peltoschema sp. (Kuschel 1990; Reid 2006) and Dicranosterna semipunctata (Chapius). The former has a much smaller body size than P. charybdis, whereas the latter has a similar body size and phenology to P. charybdis, and the first generation larvae feed on new phyllodes in December (Murray & Withers 2011). The phylogenetic relatedness of the target species to other Chrysomelidae in NZ, is an important consideration for selecting nontarget species for testing. The introduced beetle P. charybdis, belongs to the genus Paropsis, with approximately 70 species, 68 in Australia and 2 in New Guinea (Reid 2006). This is one of 11 closely related genera known in Australia as paropsines (Reid 2006; Jurado-Rivera et al. 2009). There are no native NZ paropsines, apart from the introduced pests already mentioned. The paropsines are a clade of genera in the chrysomelid subfamily Chrysomelinae, which includes more than 120 genera and 4000 species worldwide. Larvae of Chrysomelinae feed on leaves or rarely flowers. New Zealand has approximately 40 native species of Chrysomelinae in five genera: Allocharis, Aphilon, Caccomolpus, Chalcolampra and Cyrtonogetus (Reid 2006). These genera form a separate clade or clades from paropsines and are most closely related to genera in Australia, New Caledonia and South America (Reid & Smith 2004; Reid 2006; Jurado-Rivera et al. 2009; Reid et al. 2009). New Zealand also has Chrysomelinae that have socioeconomic importance as biological control agents against exotic weeds (Hayes 2007) (Table 1). Within this list are three species of Chrysolina, and one species of Gonioctena, all related to paropsines and with similar adults and larvae to P. charybdis. Furthermore several phylogenetic analyses have shown that the sister group to Chrysomelinae is Galerucinae (Reid 2014a). This is a huge subfamily with more than 11,000 species, but less than 100 in NZ. Most species have hidden larvae that feed in stems, leaf-mines or roots, and are therefore very unlikely to be available to E. paropsidis, but a few species have free-living leaf-feeding larvae New Zealand Plant Protection Society (Inc.) Refer to

4 Benefits and challenges of insect biocontrol 182 None of the latter are native to NZ (Reid 2014b), but two species are introduced biological control agents (Lochmaea suturalis (Thompson) and Agasicles hygrophila Selman & Vogt) on heather and alligator weed, respectively (Hayes 2007). Based on the phylogenetic and biological affinities it is therefore suggested that these two above-named weed biological control agents belonging to the Galerucinae should be tested against E. paropsidis (Table 1). The other native Chrysomelidae in NZ are genera in the Cryptocephalinae and Eumolpinae, neither of which are closely related to Chrysomelinae (Reid 2014a). The other introduced Chrysomelidae in NZ are species of Bruchinae (pest species), Cassidinae (1 potential pest, 1 biological control agent) and Criocerinae (4 biological control agents). Although these are phylogenetically distant from Chrysomelinae, Cassidinae and Criocerinae do contain species with external leaf-feeding larvae, for example, biological control agents from the genera Cassida, Lema and Neolema (Table 1). Because they are ecologically similar, and Chrysomelidae, a single species is included from each subfamily on the E. paropsidis non-target testing list. Preference will be given to those most easily collected, with the highest potential for spatial and temporal overlap with P. charybdis. Which representatives to choose will be considered below. Biology and phenology of NZ Chrysomelidae considered for host testing There is little information about the NZ native species of Chrysomelinae. Allocharis robusta Broun has been recorded on Veronica (Hudson 1934 as Hebe sp. cited in Spiller & Wise (1982) (Plantaginaceae) and one other species has been collected from Olearia (Compositae) (New Zealand Arthropod Collection, R.A.B. Leschen, Landcare Research, unpublished data). The apical plate of larval Allocharis may indicate that these are nocturnal larvae that burrow into stems for shelter during the day (Reid 2014b). Larvae and adults of Allocharis marginata Sharp have been reported as feeding on Veronica salicifolia (Plantaginaceae) along riverbanks (Jolivet & Hawkeswood 1995). Aphilon species are small and have been recorded on mosses and liverworts (Bryophyta) in native bush, the adults being nocturnal (Kuschel 1990). Little is known of Cyrtonogetus, but it is suspected to have nocturnal larvae, while Chalcolampra speculifera Sharp may have diurnal larvae, but nothing is known of its native habitat or hosts. Australian species of Chalcolampra have been recorded from Parahebe (Plantaginaceae) and Prostanthera (Lamiaceae) (Reid 1993). Selecting the species to test by applying the temporal and biological filters The number of phylogenetically closely related species identified above was reduced by applying the filters of temporal and biological similarity to the target. Beetles in the sub-family Chrysomelinae or the closely related Galerucinae are identified at highest risk. Phenology of the larval stages, in particular presence on leaves from late spring (November) to summer (January), is a significant limiting factor. On that basis both Chrysolina species on Hypericum are excluded as they never have larvae in early to mid-summer. Larvae of these species feed through the colder months from autumn until spring (Hayes 2007), and would be impossible to either obtain or test when E. paropsidis adults are present. Larval habitat filters exclude Neolema abbreviata (Lacordaire) and Lema basicostata Monrós as the larvae feed predominantly within host stems (Hayes 2007). Longitarsus jacobaeae (Waterhouse) and Bruchidius villosus (F.) are excluded on the basis of larvae feeding fully protected within roots or seeds, respectively. Despite its distribution in an aquatic habitat, A. hygrophila is not excluded, as it has external summer-active larvae and the host plant can invade terrestrial habitats (Stewart et al. 1999). As noted above, the list is completed by choice of one of each of the Cassidinae and Criocerinae, these being Cassida rubiginosa Müller and Neolema ogloblini (Monrós), both of which feed on plants that are common in the vicinity of eucalypts. Both have larvae that are readily available for rearing. Among the endemic Chrysomelinae, the species are selected for testing on the basis of ease of collection, ease of rearing and size. It is proposed that either Allocharis or Chalcolampra 2015 New Zealand Plant Protection Society (Inc.) Refer to

5 Benefits and challenges of insect biocontrol 183 Table 1 Beneficial weed biological control agents in the family Chrysomelidae in New Zealand, in decreasing order of phylogenetic similarity to Paropsini (Hayes 2007). Species Subfamily Target weed Larval phenology Feeding habitat Chrysolina hyperici Hypericum perforatum Autumn to spring Exposed leaf feeding larvae Chrysomelinae Chrysolina Hypericum perforatum Autumn to spring Exposed leaf feeding larvae quadrigemina Chrysomelinae Chrysolina abchasica 1 Chrysomelinae Hypericum androsaemum Spring to summer Exposed leaf feeding larvae Gonioctena olivacea Cytisus scoparius Spring Exposed leaf feeding larvae Chrysomelinae Lochmaea suturalis Calluna vulgaris Early summer Exposed leaf feeding larvae Galerucinae Agasicles hygrophila Galerucinae Longitarsus jacobaeae Galerucinae Cassida rubiginosa Cassidinae Neolema ogloblini Criocerinae Neolema abbreviata Criocerinae Alternanthera philoxeroides Spring to summer Exposed larvae feed on non-submerged leaves, aquatic habitat Senecio jacobaea Winter or summer Soil dwelling root-feeding larvae Cirsium arvense Spring and External leaf feeding, summer covered in own frass Tradescantia fluminensis Spring to summer External leaf feeding, shade, larvae covered in own frass Tradescantia fluminensis Spring to summer Internal feeding in stems, last instar may feed on leaves, shade Tradescantia fluminensis Spring to summer Internal feeding larvae in stems Cirsium arvense Early summer External leaf feeding, shade. Lema basicostata Criocerinae Lema cyanella Criocerinae Bruchidius villosus Cystisus scoparius Spring to autumn Internal seed feeding larvae Bruchinae 1 Currently in containment. species be used as the endemic species for testing. The pests T. sloanei, T. catenata and to a lesser extent, D. semipunctata, share phenology and habitat overlap to the target P. charybdis and are potential hosts. However, since resources are limited and these are pest species, they are a low priority as test species. Based on the reasoning outlined above, the species listed in Table 2 are the candidates proposed for non-target testing. The initial results of host testing will further refine this list, with additional species being added to the testing list if required, based on interim results, as is recommended by Kuhlmann et al. (2006). CONCLUSIONS The non-target list presented in Table 2 prioritises non-target species within the same subfamily to P. charybdis, the Chrysomelinae, including a species of endemic NZ beetle, if it can be obtained and cultured, as well as weed biological control agents in the sister group Galerucinae. It is hypothesised that if any non-target hosts are identified during host range testing, they will be from the most closely related species. Of lower priority, but included in the non-target species list, are one weed biological control agent from each of the less closely related Criocerinae and Cassidinae. Finally two pest beetles sharing the same niche 2015 New Zealand Plant Protection Society (Inc.) Refer to

6 Benefits and challenges of insect biocontrol 184 Table 2 Proposed non-target species list for host testing of Eadya paropsidis. Species are listed in descending order of priority. Species Subfamily Status Host Similarity to target 1 Allocharis or Chalcolampra Endemic Olearia or Veronica Same subfamily sp. Chrysomelinae 2 Chrysolina abchasica Exotic beneficial Hypericum Same subfamily Chrysomelinae androsaemum 3 Gonioctena olivacea Exotic beneficial Cytisus scoparius Same subfamily Chrysomelinae 4 Lochmaea suturalis Galerucinae Exotic beneficial Calluna vulgaris Closely related subfamily 5 Agasicles hygrophila Galerucinae Exotic beneficial Alternanthera philoxeroides Closely related subfamily 6 Neolema ogloblini Criocerinae Exotic beneficial Tradescantia fluminensis Same family as target 7 Cassida rubiginosa Cassidinae Exotic beneficial Cirsium arvense Same family as target 8 Trachymela sloanei Chrysomelinae Exotic pest Eucalyptus spp. Same subfamily, Same niche 9 Dicranosterna semipunctata Chrysomelinae Exotic pest Acacia melanoxylon Same subfamily Adjacent habitat and an adjacent habitat complete the list. The final testing may be influenced by results of the host range tests as these are conducted, as well as insect availability, phenology and resources. ACKNOWLEDGEMENTS Thanks to Ronny Groenteman, Hugh Gourlay and Richard Leschen for assistance. Co-funding of this project was provided by the MPI Sustainable Farming Fund, NZ Farm Forestry Association, South Wood Export Ltd, Carter Holt Harvey Ltd, Forest Owners Association Forest Growers Levy Trust Inc. and Scion MBIE core funding. REFERENCES Bain J, Kay MK Paropsis charybdis Stål, eucalyptus tortoise beetle (Coleoptera: Chrysomelidae). In: Cameron PJ, Hill RL, Bain J, Thomas WP ed. A review of biological control of invertebrate pests and weeds in New Zealand CAB International and DSIR, Oxon, UK. Pp Barratt BIP, Ferguson CM, McNeill MR, Goldson SL Parasitoid host specificity testing to predict field host range. In: Withers TM, Barton Browne L, Stanley J ed. Host specificity testing in Australasia: towards improved assays for biological control. Scientific Publishing, Department of Natural Resources, Brisbane. Pp de Little DW Paropsine chrysomelid attack on plantations of Eucalyptus nitens in Tasmania. New Zealand Journal of Forestry Science 19: Gourlay AH Classical biological control of Californian thistle: the New Zealand story. Weed management: balancing people, planet, profit. 14th Australian Weeds Conference: Hayes L The Biological Control of Weeds Book a New Zealand Guide. www. landcareresearch.co.nz/publications/books/ biocontrol-of-weeds-book (accessed 31 March 2015) New Zealand Plant Protection Society (Inc.) Refer to

7 Benefits and challenges of insect biocontrol 185 Hoddle MS Chapter 4. Analysis of fauna in the receiving area for the purpose of identifying native species that exotic natural enemies may potentially attack. In: Van Driesche RG, Reardon R ed. Assessing host ranges for parasitoids and predators used for classical biological control: a guide to best practice. USDA Forest Service, Morgantown, West Virginia. Pp Jolivet P, Hawkeswood TJ Host-plants of Chrysomelidae of the world: an essay about the relationships between the leaf-beetles and their food-plants. Backhuys Publishers, The Netherlands. 281 pp. Jones DC, Withers TM The seasonal abundance of the newly established parasitoid complex of the Eucalyptus tortoise beetle (Paropsis charybdis). New Zealand Plant Protection 56: Jurado-Rivera JA, Vogler AP, Reid CAM, Petitpierre E, Gómez-Zurita J DNA barcoding insect host plant associations. Proceedings of the Royal Society, Series B 276: Kean J, Suckling D, Sullivan N, Tobin P, Stringer L, Lee D, Smith G, Flores Vargas R, Fletcher J, Macbeth F, McCullough D, Herms D, et al Global eradication and response database. (accessed 31 March 2015). Kuhlmann U, Schaffner U, Mason PG Selection of non-target species for host specificity testing. In: Bigler F, Babendreier D, Kuhlmann U ed. Environmental impact of invertebrates for biological control of arthropods. CABI International, Wallingford, UK. Pp Kuschel G Beetles in a suburban environment: a New Zealand case study: the identity and status of Coleoptera in the natural and modified habitats of Lynfield, Auckland ( ). DSIR Plant Protection Report no. 3. DSIR, Auckland. 118 pp. Mansfield S, Murray TJ, Withers TM Will the accidental introduction of Neopolycystus insectifurax improve biological control of the eucalyptus tortoise beetle, Paropsis charybdis, in New Zealand? Biological Control 56: Murphy BD Biological control of Paropsis charybdis Stål (Coleoptera: Chrysomelidae) and the paropsine threat to Eucalyptus in New Zealand. PhD thesis, University of Canterbury, Christchurch, New Zealand. 150 pp. Murphy BD, Kay MK Paropsis charybdis defoliation of Eucalyptus stands in New Zealand s central North Island. New Zealand Plant Protection 53: Murray TJ, Withers TM Spread of Dicranosterna semipunctata (Col.: Chrysomelidae) in New Zealand and potential for control by intentionally introduced and invasive parasitoids. Biological Control 59: Murray TJ, Withers TM, Mansfield S Choice versus no-choice test interpretation and the role of biology and behavior in parasitoid host specificity tests. Biological Control 52: Nuttall MJ Gonipterus scutellatus Gyllenhal, gum tree weevil (Coleoptera: Curculionidae). In: Cameron PJ, Hill RL, Bain J, Thomas WP ed. A review of biological control of invertebrate pests and weeds in New Zealand CAB International and DSIR, Oxon, UK. Pp Reid CAM Description of the constricta species-group of the genus Chalcolampra Blanchard (Coloeptera: Chrysomelidae: Chryosmelinae). Journal of the Australian Entomological Society 32: Reid CAM A taxonomic revision of the Australian Chrysomelinae, with a key to the genera (Coleoptera: Chrysomelidae). Zootaxa 1292: Reid CAM 2014a. Chrysomeloidea Latreille, In: Leschen RAB, Beutel RG ed. Handbook of Zoology, Vol IV (Arthropoda: Insecta), Part 38 Coleoptera, Beetles. Morphology and Systematics. De Gruyter, Berlin. Pp Reid CAM 2014b. Chrysomelinae Latreille, In: Leschen RAB, Beutel RG ed. Handbook of Zoology, Vol IV (Arthropoda: Insecta), Part 38 Coleoptera, Beetles. Morphology and Systematics. De Gruyter, Berlin. Pp New Zealand Plant Protection Society (Inc.) Refer to

8 Benefits and challenges of insect biocontrol 186 Reid CAM, Smith K A new genus and first record of Chrysomelinae in New Caledonia (Coleoptera: Chrysomelidae). Memoirs of the Queensland Museum 49: Reid CAM, Jurado-Rivera JA, Beatson M A new genus of Chrysomelinae from Australia (Coleoptera: Chrysomelidae). Zootaxa 2207: Rice AD 2005a. The parasitoid guild of larvae of Paropsisterna agricola Chapuis (Coleoptera: Chrysomlediae) in Tasmania, with notes on biology and a description of a new genus and species of tachinid fly. Australian Journal of Entomology 44: Rice AD 2005b. The larval parasitoid guild of Chrysophtharta agricola (Coleoptera: Chrysomelidae): host parasitoid ecological and developmental interactions. PhD thesis, University of Tasmania, Hobart. 228 pp. Rice AD, Allen GR Temperature and developmental interactions in a multitrophic parasitoid guild. Australian Journal of Entomology 48: Spiller DM, Wise KAJ A catalogue ( ) of New Zealand insects and their host plants: DSIR Bulletin 231. DSIR Science Information Division, Wellington. 231 pp. Stewart CA, Chapman RB, Emberson RM, Syrett P, Frampton CMA The effect of temperature on the development and survival of Agasicles hygrophila Selman & Vogt (Coleoptera: Chrysomelidae), a biological control agent for alligator weed (Alternanthera philoxeroides). New Zealand Journal of Zoology 26: Withers TM, Allen GR, Patel VS, Satchell D, Manley G Investigating the potential of Eadya paropsidis (Braconidae) from Tasmania as a biocontrol agent for Paropsis charybdis in New Zealand. New Zealand Plant Protection 65: Withers TM, Watson MC, Watt MS, Nelson TL, Harper LA, Hurst MRH Laboratory bioassays of new synthetic and microbial insecticides to control Eucalyptus tortoise beetle Paropsis charybdis. New Zealand Plant Protection 66: New Zealand Plant Protection Society (Inc.) Refer to

9 221 Comparing traditional methods of test species selection with the PRONTI tool for host-range testing of Eadya daenerys (Braconidae) Toni M. Withers 1, *, Jacqui H. Todd 2, Belinda A. Gresham 1 and Barbara I.P. Barratt 3 1 Scion, Private Bag 3020, Rotorua 3046, New Zealand 2 The New Zealand Institute for Plant & Food Research Ltd, Private Bag 92169, Auckland Mail Centre, Auckland 1142, New Zealand 3 AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel 9053, New Zealand *Corresponding author: toni.withers@scionresearch.com Abstract A computer-based tool (PRONTI; Priority Ranking Of Non-Target Invertebrates) has been developed to assist the selection of invertebrate species for risk-assessment testing with entomophagous biological control agents (BCAs). PRONTI was used to produce a prioritised list of taxa for host-range testing with the braconid parasitoid Eadya daenerys, a potential BCA for the eucalypt pest, Paropsis charybdis. The resulting list was compared with a list developed using traditional species selection methods. Seven of the nine species on the traditional list were in the PRONTI top 20. The remaining two species (Agasicles hygrophila and Cassida rubiginosa) may not have been selected if the PRONTI tool had been used. These two species were on the traditional list because they are in the same family as the target and are considered valuable BCAs. Alternative BCAs were prioritised by PRONTI. The other 13 taxa prioritised by PRONTI were not on the traditional list: the taxa are littleknown natives in the target subfamily or sister subfamily but larvae are presumed to be root-feeders, whereas target larvae are leaf-feeders. PRONTI can support the traditional approach by providing transparent evidence to support the selection (or rejection) of nontarget species for host-range testing. Keywords host-range testing, biosafety, host-testing list, phylogeny, risk assessment. INTRODUCTION Biological control agents (BCA) proposed for release in New Zealand must first be assessed for any risks they may pose to the receiving ecosystem, particularly to non-target species that could be used as alternative hosts or prey by entomophagous BCAs (Barratt & Moeed 2005). Therefore, selecting the non-target species to test is a very important task given the large number of native and valued introduced invertebrate species in New Zealand. Traditional non-target species selection methods have been outlined by the Food and Agriculture Organization and reviewed by Kuhlmann et al. (2006) to provide biological control researchers with a sound approach to follow. In general, phylogeny plays a strong part in the species selection process (Hoddle 2004). In addition to phylogeny, species that are valued, such as beneficial BCAs and invertebrates (endemic or exotic) with cultural or aesthetic significance, are also recommended to be included in test species lists (Kuhlmann et al. 2006). Often this approach leads to a very large initial list that, for practical reasons, must be filtered by eliminating species that have disparate spatial, temporal and/or morphological characteristics from the target species. However, there is always room for interpretation when New Zealand Plant Protection 71: (2018)

10 222 assessing the phylogenetic and ecological affinities between the target host (invading pest) and each potential non-target. For instance, published phylogenies can change over time with taxonomic revisions, such as occurred with Senecio species (Compositae) endemic to New Zealand (Paynter et al. 2004), and non-targets can be overlooked as valued, such as occurred with fiddlewood (Citharexylum spinosum L.; Verbenaceae) in Australia (Manners et al. 2010). This traditional approach for drawing up a host testing list was followed in 2015 when New Zealand Eucalyptus (Myrtaceae) growers expressed the need for a BCA to target the first generation of the eucalyptus tortoise beetle pest, Paropsis charybdis Stål (Coleoptera: Chrysomelidae) (Withers et al. 2015). The candidate BCA was the solitary larval parasitoid Eadya paropsidis Huddleston and Short (Hymenoptera: Braconidae). This species has since been split into three species, following intensive molecular and field research in Tasmania (Ridenbaugh et al. 2018). The agent introduced into containment and evaluated in New Zealand is Eadya daenerys Ridenbaugh 2018 (Peixoto et al. 2018). In 2015, a non-target species list was compiled for host range testing with E. daenerys using the traditional approach for entomophagous BCAs (Kuhlmann et al. 2006); herein referred to as the traditional list. Initially, a list of the closest relatives to the target paropsine chrysomelid was made. No endemic species of paropsines occur in the New Zealand fauna so the closest related endemic species to the paropsines from the Chrysomelinae subfamily were considered, particularly those in the genera Allocharis, Aphilon, Caccomolpus, Chalcolampra and Cyrtonogetus (Coleoptera: Chrysomelidae) of which there are 40 species (Reid 2006). Species in the Galerucinae were also considered because they have been shown to be the phylogenetic sister group to the Chrysomelinae, and New Zealand has approximately 100 endemic galerucine species (Reid 2014). New Zealand also has a large number of valued introduced weed BCAs, and all 13 chrysomelids on this list were considered. This resulted in an initial list of 153 potential non-target beetles. To reduce the list to a manageable number, the list was filtered using a number of ecological attributes (Kuhlmann et al. 2006). As E. daenerys has only one generation per year present only during springtime (November- December), all species without spring-active, leaf-feeding larvae were excluded and the remaining endemic beetles with an adult body length greater than approximately 5-mm long (based on the minimum size of the smallest known host, Paropsisterna agricola (Chapuis) were prioritised. This process resulted in a list of ten non-target species to try to locate for testing with E. daenerys (Table 1). The aim of the current research was to compare the non-target species list for E. daenerys produced using the traditional method with the list produced by a new computer-based tool known as PRONTI (Priority Ranking Of Non-Target Invertebrates; (Todd et al. 2015)). The PRONTI tool consists of two parts: a Microsoft Access 2013 database (called the Eco Invertebase); and a mathematical model that uses the information in Eco Invertebase to produce a prioritised list of species for testing with the proposed BCA. Eco Invertebase is used to collate information: taxonomy; food web; biomass; ecology; anthropocentric value and testability (i.e. the ability to rear and test the species in captivity) on the invertebrate taxa found in New Zealand. It also provides data on the proposed BCA and the potential interaction between the BCA and each taxon. The model combines this information (using the selection criteria outlined in Kuhlmann et al. (2006)) and converts the data into a by which the taxa can be ranked for host range testing with the proposed BCA. PRONTI has been actively tested for its ability to improve the selection of invertebrate species for risk assessment of entomophagous BCA with two BCAs previously released in New Zealand. These were Microctonus aethiopoides Loan (Hymenoptera: Braconidae) released to control Sitona discoideus Gyllenhal (Coleoptera: Curculionidae), and Cotesia urabae Austin & Allen (Hymenoptera: Braconidae) released to

11 223 Table 1 The traditional list of non-target species for host testing with Eadya daenerys. Species are listed in order of phylogenetic relatedness to the target. Status is E = Exotic self-introduced pest, N = Native, I = Introduced beneficial biocontrol agent (BCA). Exposed larvae = larvae are known to feed externally on the leaf surface. Ranks are included for clarity, and the two native genera Chalcolampra and Allocharis are listed separately, whereas in the original list they were considered so similar that they were ranked equally. Recreated from Table 2, page 184 of Withers et al. (2015). Rank Species and Status Subfamily Host Primary reason for inclusion 1 Trachymela sloanei E Chrysomelinae Eucalyptus spp. Same subfamily Same habitat 2 Dicranosterna semipunctata E Chrysomelinae Acacia melanoxylon Same subfamily Adjacent habitat 3 Chalcolampra sp. N Chrysomelinae Olearia colensoi Same subfamily 4 Allocharis sp. N Chrysomelinae uncertain Same subfamily 5 Gonioctena olivacea I Chrysomelinae Cytisus scoparius Same subfamily, BCA 6 Chrysolina abchasica I Chrysomelinae Hypericum Same subfamily, BCA androsaemum 7 Lochmaea suturalis I Galerucinae Calluna vulgaris Sister subfamily, exposed larvae, BCA 8 Agasicles hygrophila I Galerucinae Alternanthera philoxeroides Sister subfamily, exposed larvae, BCA 9 Neolema ogloblini I Criocerinae Tradescantia fluminensis Same family, exposed larvae, BCA 10 Cassida rubiginosa I Cassidinae Cirsium arvense Same family, exposed larvae, BCA control Uraba lugens Walker (Lepidoptera: Nolidae) (Barratt et al. 2016; Todd et al. 2016). The aim of the current study was to test the PRONTI tool prior to completing an actual hostrange assessment for a BCA that was proposed for release in New Zealand. Of particular interest were whether or not: (a) PRONTI would prioritise different or additional at-risk non-target species that had not been identified using the traditional approach; and (b) if the tool provided information that may have resulted in the selection of different species from those on the traditional list (Table 1). MATERIALS AND METHODS The candidate BCA: Eadya daenerys The Eco Invertebase was populated with the following relevant biological and ecological information about the proposed BCA, E. daenerys. Eadya daenerys is a solitary larval endoparasitoid (Rice 2005) and is now known to be most commonly associated with a small number of species within the genera Paropsis and Paropsisterna (Ps.) (formerly Chrysophtharta), together known as paropsines (Peixoto et al. 2018). Eadya daenerys is patchily distributed (G. Allen and S. Quarrell pers. comm.) in Tasmania but has been repeatedly collected throughout the state since 2011, from sea level to 600 m, so is considered abundant. Adults have been collected during the months of November to January with a peak in December. Adults can fly, and larval

12 224 E. daenerys use its host for only limited dispersal (the infested host larva drops to the ground to pupate). Eadya daenerys can infest all larval instars of its paropsine hosts, which range in adult size from mm long, Ps. agricola to P. charybdis, respectively (de Little 1979; Nahrung & Allen 2004). All Eadya daenerys hosts are restricted to feeding on Eucalyptus spp., and more than one species of host can co-occur in its main habitat of mixed native Australian forests as well as commercial Eucalyptus spp. plantations in Tasmania. Model criteria and weightings Ideally, the PRONTI tool would be used to rank all invertebrate species that could be exposed to the BCA if it were released in New Zealand. In the case of E. daenerys, that would be all invertebrates found in or near Eucalyptus spp., which are very widespread in parks, gardens and plantations throughout New Zealand. Since such a list would be unreasonably large, the following ten categories were used to select a set of 127 invertebrate taxa more likely to be at risk from E. daenerys if it were released in New Zealand, and data on each were entered into the Eco Invertebase. This list included most of the 153 taxa considered using the traditional approach. However, where very little information was available for individual species within a genus (e.g., the ten species of Allocharis), the data were combined into a single entry for the genus. Although PRONTI can deal with some data gaps (see below), combining all available data into a single entry for the genus increased the certainty with which these taxa could be ranked by PRONTI. 1. Native Coleoptera most closely related to P. charybdis: we selected 16 of the 17 taxa in this category: 12 Chrysomelinae and Galerucinae taxa, plus four Chrysomelidae from other subfamilies from this category (the genus Arnomus was excluded because of the lack of available data). 2. Native Coleoptera in the same environment as P. charybdis: we selected ten of the 38 native coleopteran taxa known to feed on, or in association with, Eucalyptus spp., including some predatory beetles. 3. Valued Chrysomelidae: we included ten of the 11 established chrysomelid weed BCAs in New Zealand. 4. Other valued, non-native Coleoptera in the same environment as P. charybdis: four of the six coleopteran BCA or non-native natural enemies found in association with Eucalyptus spp. were included. 5. New Zealand Euphorinae that could be at risk of hybridisation with E. daenerys: we included ten of the 21 taxa in this category: five native species, three BCAs, and two other non-native Euphorinae. 6. Invertebrate natural enemies of P. charybdis: all four known predators, the two known parasitoids, plus three spider species found in association with Eucalyptus spp. were selected. 7. Pests of Eucalyptus spp. that could be released from competition following control of P. charybdis: we included 42 of 75 taxa in this category, including three non-native chrysomelids, 11 other Coleoptera, and 28 non-coleopteran eucalypt leaf-feeders. 8. Chrysomelid pests of other forestry trees that could be released from competition following control of P. charybdis: only Dicranosterna semipunctata on Acacia melanoxylon (Fabaccae) fits this category. 9. Coleopteran hosts of Euphorinae in New Zealand: we included 21 taxa in this category because they were already in the Eco Invertebase, including 11 weevil hosts of Microctonus spp. (Hymenoptera: Braconidae). 10. Chrysomelid pests of non-forestry plants that could be controlled by E. daenerys: all four non-native paropsine pests in this category were included. The 127 taxa were then ranked by the model that sits within the PRONTI tool by applying five selection criteria to the data in the Eco Invertebase. The criteria were: (1) the potential hazard (H) posed by E. daenerys to each non-

13 225 target taxon; (2) the potential degree of exposure (E) of each non-target to E. daenerys; (3) the hypothetical ecological impact (R & S) that may result from the exposure of the non-target to the hazard posed by E. daenerys; (4) the estimated economic, social and cultural value (V) of each non-target; and (5) the assessed ability to source each non-target and to conduct tests (T), Table 2. To enable the model to do this, each datum in the Eco Invertebase was automatically assigned a Table 2 Details of the changes to s or weights given to questions used to inform each of the criteria used in the PRONTI test with Eadya daenerys. The questions listed are only part of the data used to obtain each criterion (i.e. these changes only affected part of each ). NTS = non-target species; BCA = biological control agent. Criterion Question Reason for change Hazard (H) Exposure (E) Resilience (R) Status (S) Value (V) Testability (T) What is the phylogenetic separation between target and NTS? E. daenerys poses the greatest risk to NTS closely related to the target, so this attribute was given the highest weight in the calculation of H by multiplying all s by 2. This was also done in the test of PRONTI with C. urabae (Todd et al. 2016). Does the NTS occur These attributes were given the highest weight (multiplied by 2) in same community or in the calculation of E when PRONTI was tested with M. habitat as the target? aethiopoides because of the risk posed to NTS in the same habitat as the target. This was a lower risk for NTS from E. daenerys because this agent is not known to attack unrelated species in the same habitat in Australia, so the multiplier was removed. Five questions on NTS mobility, abundance, host range, dispersal and phenological overlap Is the NTS rare or are there many documented foodweb links? Is the non-target endemic or rare? Can the non-target be collected, reared, or does an existing protocol for rearing exist? Reducing the weight of attributes used to calculate E also reduced the maximum risk (i.e., H E). In Equation 1, the NTS risk is modified by the R (i.e. the ability of the NTS to mitigate the risk). If the maximum risk is reduced, the maximum resilience must also be reduced so that it doesn t have too much weight in the model. Consequently, the s for each of these attributes were reduced in this test of PRONTI by dividing them by 2. The S indicates how important each NTS is to the ecosystem (i.e., NTS with large biomass and many links to the foodweb are more important ). In previous tests of PRONTI, rare NTS were assigned a biomass of per m 2 ; however, this was found to reduce the NTS status unfairly. Thus, in this test, rare NTS were assigned a biomass of 0.01 per m 2. Also, NTS that have been well studied may have many known foodweb links and obtain a much higher status than other NTS. Consequently, foodweb s were all divided by 2 to reduce their weight in the calculation of S. Rare or endemic NTS often obtain a low S because of their predicted low biomass and few known foodweb links. However, these species are highly valued, so questions on rarity and endemicity were multiplied by 3 in the calculation of V in this test of PRONTI. Criteria S, V and T were designed to be approximately equal because each was considered an equally important criterion for selecting NTS. Examination of these s in this test showed that the average T was higher than the average S and V s. In previous tests of PRONTI, the attributes used to measure T were multiplied by 2, so these multipliers were removed in this test of PRONTI to bring T in line with S and V.

14 226, ranging from 0 to 10, with 10 assigned to those attributes that were most informative to the selection criteria. For example, species known to feed on eucalypt leaves were assigned a of 10 for criterion 2 (exposure), while species feeding on other parts of eucalypt trees, or on other plant species, were assigned lower s. A number of different pieces of data in the Eco Invertebase were used to inform each of the five criteria. Where data were unknown for a taxon, the middle of five was always assigned, and this was then used to calculate the amount of uncertainty in each species ranking (see Todd et al. (2015) for more details). With each test of PRONTI, the attributes of the agent, the characteristics of the agent s hosts, and the context into which the agent would be released were examined, and changes were made where the s or the weights given to certain attributes were more or less relevant to a selection criterion. For example, where an agent was only known to attack species that were closely related, the data representing that relationship were given a higher hazard weighting, whereas for agents known to attack species in a particular feeding niche, these data were given the highest weighting. Changes made to s and weights for E. daenerys are given in Table 2. To produce the prioritised list of species, several questions and therefore several s were combined to provide a total for each criterion. The s for each criterion were then combined using the following equation: PRONTI = [(H x E)/R ] (S+V+T) Equation 1 Where H = hazard (criterion 1); E = exposure (criterion 2); R = resilience (ability of the species to mitigate the risk; criterion 3); S = the status of the species in the receiving environment (criterion 3); V = the value of the species (criterion 4); and T = the testability of the species (criterion 5). More details of s, weights and criteria and how they are applied and calculated in the PRONTI tool can be found in Todd et al. (2015). RESULTS AND DISCUSSION A number of diagnostic tools were used to check the model was working correctly and that data had been entered consistently. Firstly, dummy high, low and medium risk species were used to check that the s were being applied appropriately in the model and were being ranked as expected (Todd et al. 2015; Barratt et al. 2016). Secondly, the s obtained by the non-target species for each of the five criteria were examined to ensure the data gaps (i.e. all the unknown attributes assigned a of five) were not having undue influence on the s for any of the criteria, or on the final PRONTI s. These attributes may be assigned higher or lower s when known, so it is important to ensure that taxa with many unknown attributes do not obtain the highest or lowest for any criterion. The top 25 non-target species ranked using the PRONTI tool are provided in Table 3, along with the rank assigned using the traditional method for test species selection. The top 20 taxa on the list produced by the PRONTI tool (herein referred to as the PRONTI list ) are different to those on the traditional list (Table 1), although the lists have seven taxa in common. Deciding which taxa to test from the PRONTI list requires interrogation of the data in the table: the PRONTI tool was developed as a decision-support system such that the information in the list should be used to support decisions on the taxa to undergo risk assessment with the BCA. For instance, it may be decided that taxa with low uncertainty in their ranking (e.g. less than 50%), and where the hazard is high (e.g. a hazard greater than 75) should be selected, and only one species from each genus should be included in the test species list. Using these criteria, the following ten Chrysomelidae could be selected from the PRONTI list for testing with E. daenerys: Gonioctena olivacea, Trachymela spp., Chaetocnema spp., Chrysolina abchasica, Aphilon spp., Pleuraltica cyanea, Allocharis spp., Longitarsus jacobaeae, Alema spp., Chalcolampra speculifera. Whichever species are selected, the data in the table can be used to justify those decisions in any application to release the BCA submitted to the New Zealand