D.T. Briese a, A. Walker a, W.J. Pettit a & J.-L. Sagliocco a a CSIRO Entomology and CRC forweed

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1 This article was downloaded by: [CSIRO Library Services] On: 23 June 2015, At: 21:01 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Biocontrol Science and Technology Publication details, including instructions for authors and subscription information: Host-specificity of Candidate Agents for the Biological Control of Onopordum spp. Thistles in Australia: An Assessment of Testing Procedures D.T. Briese a, A. Walker a, W.J. Pettit a & J.-L. Sagliocco a a CSIRO Entomology and CRC forweed Management Systems, GPO Box 1700, Canberra, 2601, Australia Published online: 28 Jun To cite this article: D.T. Briese, A. Walker, W.J. Pettit & J.-L. Sagliocco (2002) Host-specificity of Candidate Agents for the Biological Control of Onopordum spp. Thistles in Australia: An Assessment of Testing Procedures, Biocontrol Science and Technology, 12:2, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis.

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3 Biocontrol Science and Technology (2002) 12, Host-speci city of Candidate Agents for the Biological Control of Onopordum spp. Thistles in Australia: an Assessment of Testing Procedures D. T. BRIESE, A. WALKER, W. J. PETTIT and J.-L. SAGLIOCCO CSIRO Entomology and CRC for Weed Management Systems, GPO Box 1700, Canberra 2601, Australia (Received 17 April 2001; returned 25 May; accepted 17 August 2001) This paper describes a series of host-speciwcity tests carried out to evaluate the safety of eight insects for release in Australia as biological control agents of Onopordum spp. thistles; Larinus latus L., Lixus cardui Ol. and Trichosirocalu s briesei Alonso-Zarazaga & Sanchez-Ruiz (Coleoptera: Curculionidae ), Tephritis postica Loew and Urophora terebrans Loew (Diptera: Tephritidae), Botanophil a spinosa Rondani (Diptera: Anthomyiidae), Tettigometra sulphurea Mulsant & Rey. (Hemiptera: Fulgoroidae ) and Eublemma amoena (Hbn.) (Lepidoptera: Noctuidae). Preliminary tests were Wrst carried out agains t key test plants in the country of origin to determine whether it was worth proceeding with formal testing under quarantine conditions in Australia. In the formal testing procedure, the test plant list was based solely on phylogeneti c relationships. The life-stage of each insect tested was selected with respect to its biology and behaviour, e.g. only oviposition was tested for insect species with immobile endophagou s larvae, while larval host utilization was tested for those with mobile larval stages. The initial laboratory experiments for each species used were caged no-choice tests, as conwnement without choice can elicit extreme behavioural responses, making negative results extremely robust. Where positive results occurred, insects where re-exposed to the plant, but this time given a choice between the target and non-target plants. The results of these tests, plus data from open-weld preliminary tests in the country of origin, helped interpret results and determine the risk posed by each candidat e biological control agent to non-target plant species. Following submission, all eight candidates were approved for release by the Australian plant biosecurity and conservation authorities. Based on a comparison of the eight species tested, it is recommended that host-speciwcity testing be kept Xexible and pragmatic, rather than moving towards a formulaic risk assessment procedure. Moreover, given the improved state of knowledge of plant phylogenie s and the evolution of host utilization, it is time to base testing procedures purely on phylogeneti c grounds, without the need to include less related test species solely because of economic or conservation reasons. Keywords: host-speciwcity testing, weed biologica l control, Onopordum spp., Larinus latus, Lixus cardui, Trichosirocalus briesei, Tephritis postica, Urophora terebrans, Botanophila spinosa, Tettigometra sulphurea, Eublemma amoena Correspondence to: D. T. Briese, CSIRO Entomology and CRC for Weed Management Systems, GPO Box 1700, Canberra 2601, Australia. ISSN (print)/issn (online)/02/ DOI: / Taylor & Francis Ltd

4 150 D. T. BRIESE ET AL. INTRODUCTION The host-speci city testing of weed biologica l control agents is a critical procedure, carried out to evaluate the risk posed to non-target plant species should they be introduced into a new range where the target weed has become invasive. This testing has become a topic of ongoing debate (see Wapshere, 1989; Harris & McEvoy, 1995; Marohasy, 1998; Withers et al., 1999). The two core issues in this debate are the selection of test plants used to determine the host range and the design of experiments used to measure agent response to these test plants. Most test plant lists are still based on the centrifugal phylogenetic strategy of Wapshere (1974), which prioritizes test plants in order of their taxonomic relatedness to the target species. However, as a safeguard, this strategy also recommends testing cultivated plants that: (i) may not be related, but that have poorly known natural enemy guilds; (ii) have not been exposed to the agent; or (iii) are attacked by organisms closely related to the agent under test. Recent evidence indicates that host-shifts in lineages of specialist phytophagous insects are strongly linked to the evolution of host-plant lineages, and that such insects show a strong phylogenetic conservatism of host associations (Mitter et al., 1991; Futuyma et al., 1995; Kopf et al., 1998; Kelley & Farrell, 1998). There has also been an enormous increase in the use of molecular data to determine plant taxonomic relationship s (e.g. Downie & Palmer, 1992; Olmstead et al., 1992; Michaels et al., 1993; Angiosperm Phylogenetic Group, 1999). Molecular phylogenies have clari ed both plant aynities (Wagenitz, 1997) and are generally indicative of relationships in plant chemistry (Grayer et al., 1999). Greater knowledge of the evolution of host-choice in specialist insects, greater con dence in the determination of plant phylogenies, and the experience of host-speci city testing over the last 25 years suggest that there is no longer a need for non-phylogenetically based `safeguard categories. Over 200 agents from a taxonomically diverse range of weeds have been tested since Wapshere (1974) rst proposed this strategy (see Julien & GriYths, 1998), and all known examples of agents colonising non-target plants involve either congeneric species or taxa from closely related genera (e.g. Pemberton, 2000). Host-speci city testing of biologica l control agents is an area of ongoing analysis and debate (see Heard & van Klinken, 1998; Sheppard, 1999; van Klinken, 2000). DiVerent authors have discussed the bene ts and drawbacks of conducting caged no-choice tests (Hill, 1999), tests involvin g choice between target and non-target plants (Edwards, 1999) and open- eld testing (Briese, 1999). Recent reviews of host-speci city testing have emphasized an understanding of insect behaviour in designing and interpreting tests, and have pointed out the possibilities of false results where this is not done (Marohasy, 1998; Heard, 2000; Withers et al., 2000). Recently, more formalized risk assessment procedures, in which risk is quanti ed for all test plants for both the probabilit y of establishment (e.g. oviposition choice) and the consequences of establishment (e.g. feeding and development), have also been recommended (Wan & Harris, 1997; Withers, 1999). The host-speci city testing of candidat e agents for the biologica l control of Onopordum spp. thistles has provided an opportunit y to examine these issues. Species of this genus are considered severe exotic pasture weeds in southern Australia (Parsons & Cuthbertson, 1992). Following extensive exploration in their native range in Mediterranean Europe (Briese et al., 1994), eight insect species were selected as candidate biologica l control agents (Briese et al., 2002). This paper presents the results of a range of tests used to determine the host-speci city of these candidat e agents, in order to seek approva l for their release against Onopordum spp. in Australia, and discusses their risk to non-target species. The data are also used to examine whether the selection of test plants can be safely based solely on phylogenetic relationship s of species to the target plant, and whether host-testing methodology should move toward more formalized risk-assessment procedures or adopt a more pragmatic and exible approach to the types of test used and life-stage of insect tested.

5 HOST SPECIFICITY OF CANDIDATES AGAINST THISTLES 151 MATERIALS AND METHODS Target Weed The genus Onopordum is a Eurasian taxon of biennial or facultatively perennial monocarpic herbs, including 13 species that occur in Europe, mainly in the Mediterranean basin (Amaral Franco, 1976). Three species from this area, O. acaulon L., O. acanthium L. and O. illyricum L., have become widely naturalized in southern Australia and are weedy in pastures (Briese et al., 1990). O. acanthium and O. illyricum are the two most serious weeds and are the main focus of biologica l control. These two species have hybridized freely since their introduction into Australia and many weedy population s consist solely of plants with varying degrees of hybridizatio n (O Hanlon et al., 1999). Plants used in individua l host-speci city tests described below comprised individual s of both parent species and their hybrids. This did not compromise the testing, as eld surveys in Europe indicated that the candidate agents used both species as host-plants (Briese et al., 1994), and there are no non-target species within this genus in Australia. Hence, the target plants used in tests are hereafter referred to as Onopordum spp. Candidate Biological Control Agents The eight insect species tested included three species whose larvae feed internally in the capitula of the thistles and destroy seed, namely the weevil, Larinus latus L. (Coleoptera: Curculionidae) and the seed ies, Tephritis postica Loew and Urophora terebrans Loew (Diptera: Tephritidae). Adults of the weevil also feed on leaf tissue, whereas adults of the two y species are nectar feeders. Two insect species that attack the owering stems were also tested; the weevil, Lixus cardui Ol. (Coleoptera: Curculionidae), adults of which feed on leaf tissue and larvae of which mine the stem, and the planthoppe r, Tettigometra sulphurea Mulsant & Rey. (Hemiptera: Fulgoroidae), all stages of which are external and suck sap from the cells of the leaves and stems. Finally, three insect species that attack the rosette stage of Onopordum spp. were also tested; the weevil, Trichosirocalus briesei Alonso- Zarazaga & Sanchez-Ruiz (Coleoptera: Curculionidae), and the y, Botanophila spinosa Rondani (Diptera: Anthomyiidae) both have larvae that feed on meristem tissue in the crown of the rosette, while larvae of the moth, Eublemma amoena (Hbn.) (Lepidoptera: Noctuidae) mine the leaf petioles and can bore down into the central tap root. T. briesei adults feed on leaves of the host plant, while adults of the y and moth are nectar feeders. Details of the biology and life-cycles of these species are given in Briese et al. (2002). Field observations and literature records indicated that these candidate agents for biological control of Onopordum spp. were either con ned to hosts within the genus Onopordum or, at the broadest, within the tribe Cardueae of the family Asteraceae (Briese et al., 1994). Host-speci city Test List The approved test plant list for candidate biologica l agents of Onopordum spp. thistles (Table 1) was partly based on a list previously approve d by Australian quarantine authorities for testing the host-speci city of insects introduced for the biological control of Carduus nutans, another species of carduine thistle (e.g. Woodburn, 1993). The test list included only members of the family Asteraceae, a large natural family that is well de ned by specialized oral characteristics and distinctive secondary chemistry (Cronquist, 1981; Michaels et al., 1993; Gustafsson 1996). Recent studies have added signi cantly to an understandin g of relationship s among taxa within the Asteraceae. For example, the tribe Carduineae, which contains Onopordum, has recently been elevated to subfamily status, Carduoideae, by Bremer (1996). This emphasizes its distinctness from the subfamilies Cichorioideae and Asteroideae, to which most native Australian species of Asteraceae belong. Morphologica l phylogenies have been determined for the tribe Cardueae to which the genus Onopordum belongs (Petit, 1997), as well as for the subfamilies Cichorioideae (Karis et al., 1992) and Asteroideae (Karis, 1993). In addition,

6 152 D. T. BRIESE ET AL. TABLE 1. Species of the family Asteraceae approved for host-speci city testing of candidate biological control agents for Onopordum spp. thistles in Australia (subfamilies according to Bremer, 1996) Subfamily Carduoideae Tribe Cardueae Sub-Tribe Carduinae Cynara scolymus L. E Hemistepta lyrata Bunge N Silybum marianum (L.) Gaertner W Carduus nutans L. W Cirsium vulgare (Savi) Ten. W Carthamus lanatus L. W Sub-Tribe Centaurinae Carthamus tinctorius L. E Stemmacantha australis (Gaudich.) Sojak N Subfamily Cichoroideae Tribe Lactuceae Microseris scapigera (Cunn.) Schultz-Bip. N R Tribe Vernonieae Vernonia cinerea Less. N Subfamily Astereae Tribe Helianthineae Glossogyne tenuifolia (Labill.) Less. N Melanthera bixora (L.) Willd. N Helianthus annuus L. E Helianthus tuberosus L. E Tribe Astereae Calotis scapigera Hook. N Tribe Inuleae Bracteantha bracteata (Vent.) Andrews N Tribe Senecioneae Senecio linearifolius A. Rich. N R Tribe Anthemideae Centipedia minima (L.) Braun & Asch. N R Tribe Arctoteae Cymbonotus preissianus Steetz N Ungrouped Tribe Mutisieae Trichocline spp. N R N Australian native plants R Replaced by species in the same tribe for some tests E Economic plant W Weedy species (globe artichoke) (variegated thistle) (nodding thistle) (spear thistle) (savron thistle) (sazower) (Austral corn ower) (Yam daisy) (Native cobbler s peg) (Sun ower) (Jerusalem artichoke) (Tufted burr daisy) (Golden everlasting) (Mountain cotula) (Austral bear s ear) several molecular phylogenies have been determined for relevant sections of the Asteraceae (e.g. Kim et al., 1992; Jansen & Kim, 1996). Bremer (1996) has summarized the phylogeneti c patterns within tribes of the Asteraceae, and these have been combined with Petit s (1997) treatment of the Cardueae to show the phylogeneti c relationships of the test plants used for testing the candidate agents of Onopordum spp. (Figure 1). The key criterion for inclusion in the test plant list was the degree of phylogeneti c relatedness to the genus Onopordum. Economic or indigenou s plants formed part of these selected taxa where possible, but not by virtue of any other special status. As there are no native Australian members of the genus Onopordum, and all introduced Onopordum spp. are considered to be weedy, speci city to the level of genus would pose no risk to non-target plants. Test plants included the sister genus Cynara, followed by representatives of the next most related clade, comprising all other genera of the subtribe Carduineae, and two species of the subtribe Centaurineae, the next removed clade (Figure 1). Thus, following Wapshere

7 HOST SPECIFICITY OF CANDIDATES AGAINST THISTLES 153 FIGURE 1. Phylogeny of representative Asteraceae taxa (subfamilies, tribes, sub-tribes and genera) used for host-speci city testing of candidate agents for the biological control of Onopordum spp. (based on phylogenies published by Bremer, 1996, and Petit, 1997). Numbered circles indicate clades with increasing degrees of separation from Onopordum spp. See Table 1 for more details of individual species used. (1974), testing was concentrated on possible hosts amongst the most closely related taxa. These test plants included the two species of some economic importance in Australia, Cynara scolymus (globe artichoke) and Carthamus tinctorius (sazower), and one of the two Australian native species, Stemmacantha australis. The only other Australian carduine species, Hemistepta lyrata, had a diverent biogeographi c range to that of the target weeds and was so rare that seeds were unobtainable. Hence it was not included in the test list. In Australia, most speciation has occurred in other tribes of the Asteraceae. The test list therefore included one representative native species from each of these tribes, belonging to clades that were increasingly distant from Onopordum (see Figure 1), to verify that more distantly related taxa were not at risk (Table 1). Types of Host-speci city Test Used For seven of the eight candidate agents, preliminary host-testing against key test plants was carried out in the native range (France or Greece) to determine whether importation into Australia and detailed quarantine screening were warranted. The key test plants were species belonging to the same phylogeneti c clade as Onopordum, the subtribe Carduineae, and its sister clade, the subtribe Centaureineae, i.e. the non-target species most at risk of attack. One insect species, the weevil Trichosirocalu s briesei, was not given preliminary testing as it had been originally identi ed as a biotype of T. horridus, a species previously subject to rigorous host-testing procedures. Preliminary screening always involved choice tests, in which the agent was exposed to both target and non-target plants, either in the open- eld (see Briese et al.,1995) or caged in a glasshouse (see Vitou et al., 2001). Results of preliminary

8 154 D. T. BRIESE ET AL. open- eld choice tests were also used to help interpret subsequent data from testing in quarantine conditions. Formal testing against the approved test plant list was carried out between 1990 and 2000 in the CSIRO Black Mountain Quarantine Facility, Canberra, Australia, using population s of the candidate agents that had been eld collected in their native range and imported from the CSIRO European Laboratory, Montpellie r, France. The insects were tested sequentially according to the order of release determined by the Onopordum biological control strategy (see Briese et al., 2002); L. latus during , L. cardui during , T. postica during , T. sulphurea during , T. briesei during 1996, E. amoena and B. spinosa during and U. terebrans during In order to avoid unnecessary assays, a pragmatic approach has been taken toward the types of tests used and the life-stages of the agents tested. The insect life-stage initially tested depended on the biology and behaviour of individua l agents (see also Sheppard, 1999). Where the immature stages were immobile, such as endophagou s coleopteran and dipteran larvae, testing was restricted to ovipositio n choice by adults and subsequent measurement of survival to adulthood, if eggs were laid on non-target plants. In the case of mobile larvae or nymphs, the behaviour of these stages was also tested. Adult feeding tests were not necessary for those agents with nectar- or pollen-feeding adults, such as the dipteran and lepidopteran species. The agents were exposed initially to a replicated series of no-choice oviposition and/or feeding experiments involvin g pre-test controls, exposure to test plants and post-test controls. Such sequential no-choice tests have been found by Withers et al. (2000) to overcome any time-dependent change in agent responsiveness to a test plant, provided there is suycient length of exposure to the test plant (which in the case of their study with a leaf-feeding beetle was more than two days). This reduces the risk of false negative results (when the test fails to indicate a plant that can actually serve as a host), though caged no-choice tests may still produce false positive results (when the test indicates that the insect uses a plant as a host when it in fact does not) by forcing host-discrimination behaviour to extreme limits through containment of the agents. As a consequence, results indicating no response to a non-target plant under caged conditions are very robust because any errors will be on the conservative side. Hence, they give con dence to regulators and the public (Hill, 1999). Positive results, however, should be treated with caution, as they may not accurately re ect eld behaviour where the agent has the choice to leave the non-target plant (Harris & McEvoy, 1995). Hence, when no-choice tests produced ambiguous results or indicated that there may be some risk, choice tests, in which agents were presented with target and nontarget plants, were carried out to clarify agent behaviour. The testing sequence for the diverent classes of agent is shown in Figure 2. Experimental Design and Measurements Initial no-choice adult ovipositio n tests were carried out for all eight agents in mesh cages ( cm). Prior to the no-choice tests on non-target plants, the ability of the females to lay eggs was con rmed by exposing them to caged host Onopordum spp., then transferring them for 3-4 days to cages containing the test plants at the appropriat e stage of growth. The numbers of eggs laid were counted after this period and, in the case of those insects whose adults also ate leaf tissue, adult feeding damage was measured. Feeding damage on test plants was rated on a scale of 0 to 4 relative to that observed previously on the target Onopordum spp., where 0 5 no damage, 1 5 exploratory feeding (< 5% of that on Onopordum spp.), 2 5 some feeding, but substantially less (6-20%) than on Onopordum spp., 3 5 moderate feeding (21-50% of that on Onopordum spp.) and 4 5 feeding equivalent to that on Onopordum spp. The insects were then transferred back on to Onopordum spp. for a similar period to ensure that they were still capable of producing eggs. Pollen and honey solution were provided for adults of those species that required it as a food source during ovipositio n tests. Between four and six replicates were carried out for each test plant,

9 HOST SPECIFICITY OF CANDIDATES AGAINST THISTLES 155 FIGURE 2. Testing sequence for candidate biological control agents of Onopordum spp. thistles. For each species the sequence continued until results were no longer ambiguous. Adjacent boxes indicate concurrent tests (see text for details). depending on the insect tested. Only those replicates in which egg-laying occurred prior to and after exposure to the test plant were considered valid. In the case of U. terebrans, it was necessary to repeat the test using two replicates of a large walk-in cage ( m), as ovipositio n was inhibited, even on the host Onopordum spp., when con ned in small cages. Where any level of ovipositio n or feeding was observed in the no-choice tests, choice tests were subsequently carried out. These tests initially involved exposing adults of the candidate agents to both the target Onopordum spp. together with three non-target species in mesh cages ( cm). Adults were left caged for up to seven days, depending on species, after which eggs laid on individual plants were counted and any feeding damage rated as previously described. All choice tests were replicated three times. L. latus, T. briesei and T. sulphurea were subjected to these tests, and the latter species was also re-tested in a much larger cage ( m), which permitted greater mobility for the insects and was behaviourally less constraining. This was because T. sulphurea still laid eggs on non-target plants under choice conditions, and clari cation was needed on the evect of cage-size and hence insect mobility on non-target oviposition. The remaining ve agents did not proceed to choice testing as the results of the no-choice tests were unambiguous. One candidate agent, the planthoppe r, T. sulphurea, has highly mobile immature stages. Therefore, larvae were also subjected to a no-choice test. Egg clusters or newly emerged nymphs were placed directly onto the test plant and survival monitored at regular intervals.

10 156 D. T. BRIESE ET AL. This was compared against survival of eggs and nymphs placed on Onopordum spp. at the same time. Another agent, the moth E. amoena, has larvae that normally feed internally on the host plant on which they hatch, but which can move between plants if crowding or a deterioration in plant quality occur. A choice test was therefore set up for migrating E. amoena larvae, in which three replicates of each test plant plus an equal number of unattacked Onopordum spp. plants were placed around potted Onopordum spp. rosettes that contained high densities of developing E. amoena larvae. After one month, just prior to pupation of the larvae, all plants were dissected to determine the levels of colonization and feeding damage by migrating larvae. RESULTS Preliminary Testing in France Host-speci city testing in Europe was carried out both in the eld, with complete agent mobility, and in the laboratory under caged conditions. The results of open- eld tests have been discussed in detail by Briese et al. (1995), while Vitou et al. (2001) provide more information on laborator y testing. In order to compare these data with those of subsequent testing of a broader range of species under quarantine conditions in Australia, the results were standardized against the agent s performance on Onopordum spp. For replicates of each test and insect species, data obtained from all plants were transformed by dividing the result for the test plant by the result for control Onopordum spp. multiplied by 100. Thus Onopordum was scored at 100 for each test and results for other test plants received a relative value (0-100). An overall performance index was calculated as the mean of the results for each replicate. The relative performances of the candidate agents on key plant species in the tribe Cardueae are shown in Table 2. Laborator y testing indicated that B. spinosa, E. amoena and U. terebrans were restricted to the genus Onopordum under choice conditions. The data indicated possible diverences in preference by all three insects for O. acanthium and O. illyricum (Table 2). The signi cance of this could not be tested but, in the case of B. spinosa, sampling of natural population s suggested an association with O. acanthium (Briese et al., 2002). The open- eld tests were equally conclusive for L. latus, L. cardui and T. postica. The two weevil species showed little diverence in attack levels between the two Onopordum species, but T. postica again showed a preference for O. acanthium. The higher ovipositio n rates by the two capitulum-attackin g ies on O. acanthium may be a re ection of the higher number of capitula produced by that species relative to O. illyricum. The only agent to show ambiguous results was the sapsucking bug, T. sulphurea, which also laid eggs on Cynara spp., though nymphs hatching from these did not survive to adulthood and there was a strong preference for Onopordum spp. over Cynara spp. (Table 2). The results indicated that all species showed a very high degree of speci city and that the evort of importing them into Australia for further testing in a quarantine facility against a formal list of test plants approved by Australian plant biosecurity oycials would be worthwhile. Quarantine Testing in Australia A total of 19 tests were performed on eight agents. Data were collected in both quantitative (e.g. egg counts) and semi-quantitative (e.g. rating of feeding damage) form, and absolute results varied considerably between agent species tested due to diverences in their biology. To facilitate comparisons between species and between test plants, the results were standardized against the agent s performance on Onopordum spp., as described above. These scores are shown in Table 3, which summarizes the results of quarantine host-speci city testing for all candidate biologica l control agents. Under no-choice cage tests in quarantine, the three dipteran species, T. postica, B. spinosa and U. terebrans, showed complete discriminatio n in ovipositio n behaviour, with no eggs

11 HOST SPECIFICITY OF CANDIDATES AGAINST THISTLES 157 TABLE 2. Preliminary testing of candidate agents in Montpellier, France, for the biological control of Onopordum spp. thistles (all test result scores relative to the highest measurement which is set at 100Ð see text for details) Agent species Larinus latus Lixus cardui Tephritis postica Tettigometra sulphurea Botanophila Eublemma Urophora spinosa amoena terebrans Life-stage tested a Oviposition Oviposition Oviposition Oviposition Immature Oviposition Oviposition Oviposition Test type b C (open) C (open) C (open) C (lab) C (open) C (open) C (lab) C (lab) C (lab) Tribe Cardueae Sub-Tribe Carduinae Onopordum acanthium Onopordum illyricum Cynara cardunculus Cynara scolymus Carduus nutans Carduus pycnocephalus 0 Cirsium vulgare Silybum marianum 0 Sub-Tribe Centaurinae Carthamus lanatus Carthamus tinctorius a Oviposition 5 adult choice of host for oviposition; Immature 5 survival of immatures to adulthood. b C (open) 5 open- eld choice test; C (lab) 5 Laboratory-based choice test.

12 158 D. T. BRIESE ET AL. being laid on species outside the genus Onopordum (Table 3). As none of these species have mobile larvae, further tests were not needed to demonstrate their speci city. The moth, E. amoena, did lay a few eggs on one non-target plant, the carduine thistle, C. vulgare, and one larva developed to adulthood, but when the mobile larvae were provided a choice test, they colonized only Onopordum spp. (Table 3). This indicates that E. amoena presents a very low risk to non-targe t species within the subtribe Carduinae (all of which are exotic weeds in Australia) and no risk to other non-target species. Subsequent exposure of this insect to both C. vulgare and Onopordum spp. during mass-rearing in a large walk-in cage con rmed that C. vulgare was not attacked under choice conditions. Adults of the three weevil species, L. latus, L. cardui and T. briesei, did feed on the foliage of both subtribes of the Cardueae thistles, including the Australian native, S. australis, under no-choice conditions. L. latus and L. cardui also made some explorator y feeding holes on a few other Asteraceae (Table 3). Moreover, under no-choice conditions, both L. latus and T. briesei laid a relatively small number of eggs on some of the Cardueae test plants. In the case of L. latus, eggs were also laid on the cages containing the non-target plants, suggesting that ovipositio n behaviour was not normal under these conditions. While all hatching L. latus larvae developed to some extent in the capitula of the carduine thistles, only one completed development to adulthood, in a capitulum of S. marianum (Table 3). Subsequent choice tests in quarantine showed that L. latus exhibited either no or much reduced feeding and laid no or only a few isolated eggs on these species (Table 3), while data from the preliminary open- eld tests in France showed no attack on Cynara, the most closely related non-target genus (Table 2). Neither S. australis nor more distantly related Asteraceae taxa were attacked by L. latus under quarantine choice tests (Table 3). Only the planthoppe r, T. sulphurea, showed indiscriminate feeding and ovipositio n behaviour in no-choice tests. Adults were much more discriminating in oviposition under choice conditions, though some eggs were still laid on non-target plants (Table 3). No-choice feeding experiments, involving the transfer of nymphs, indicated that they could survive for varying periods of time on some non-targe t species, though development to adulthood was successful on only the carduine thistle, C. scolymus. Lower survival and longer development time on this species relative to Onopordum indicated that it was an inferior host plant for T. sulphurea. DISCUSSION The tests described here successfully addressed the safety of candidate agents for introduction into Australia for the biological control of Onopordum spp. thistles. Five of the agents demonstrated complete restriction to the target genus Onopordum for successful completion of their life-cycle. Three species, the noctuid moth, E. amoena, the weevil, L. latus, and the planthopper, T. sulphurea, were found to be able to develop to adulthood, each on one closely related species of non-target thistles, though survival and growth were low relative to that on Onopordum spp. However, under choice conditions in quarantine tests, mobile E. amoena larvae did not colonize non-target plants, which suggests that there is very little risk posed to other plant species. Open- eld experiments in the native range showed that L. latus was restricted to Onopordum spp., and indicated a strong preference by T. sulphurea for Onopordum spp. with no survival of nymphs on other carduine thistles. Moreover, a naturally occurring populatio n of T. sulphurea laid eggs only on Onopordum spp. and not on neighbouring Cynara spp. during an open- eld test in Greece (Briese et al., 1995). One risk issue peculiar to sap-sucking insects, such as T. sulphurea, is that they could act as vectors of non-persistent plant viruses when probing non-host plants, due to mechanical infection of the stylus or foregut (Nault & Ammar, 1989). However, considering the size of the Australian hemipteran fauna as a whole, it was considered that the introduction of one additional oligophagou s species would contribute marginally to the general risk of virus transmission and, even if local numbers were large, would not necessitate changes to existing control practices for the reduction of plant virus transmission (see Briese, 1989).

13 HOST SPECIFICITY OF CANDIDATES AGAINST THISTLES 159 TABLE 3. Detailed host-speci city testing in quarantine of candidate agents for the biological control of Onopordum spp. thistles (all test result scores relative to the highest measurement which is set at 100Ð see text for details) Species Tephritis Urophora Botanophila Eublemma amoena Larinus latus Lixus cardui Trichosirocalus briesei Tettigometra sulphurea postica terebrans spinosa Larval survival a Ovi- Ovi- Ovi- Ovi- Oviposition Adult feeding Oviposition Larval Adult Ovi- Adult feeding Oviposition Oviposition Larval position position position position survival feeding position survival Test type b NC NC NC NC NC C NC C NC C NC NC NC NC C NC C NC C NC Tribe Cardueae Sub-Tribe Carduinae Onopordum spp Cynara scolymus Cynara cardunculus Silybum marianum Carduus nutans Cirsium vulgare Sub-Tribe Centaurinae Carthamus lanatus Carthamus tinctorius Stemmacantha australis Tribe Heliantheae Glossogyne tenuifolia Helianthus annuus Helianthus tuberosus Melanthera bixora Tribe Astereae Calotis scapigera Tribe Inuleae Bracteantha bracteata Tribe Senecioneae Senecio gunnii Senecio linearifolius Senecio odoratus 0 0 Tribe Anthemideae Centipedia cunninghamii Centipedia minima Tribe Arctoteae Cymbonotus preissianus Tribe Lactuceae Microseris lanceolata Microseris scapigera Tribe Mutisieae Gerbera sp Trichocline spathulata Tribe Vernonieae Vernonia cinerea a Lifestages tested were: Adult adult feeding; Oviposition adult choice of host for oviposition; Immature survival of immatures to adulthood. b NC no-choice test; C choice test between target weed and non-target plants.

14 160 D. T. BRIESE ET AL. Following consideration of application s made to Biosecurity Australia and Environment Australia, all eight agents were approved for eld release. All except the planthoppe r, T. sulphurea, were eventually released between 1992 and This insect was not released due to concerns that ants attracted to planthoppe r colonies might interfere with other biological control agents (see Briese et al., 2002). Two of the agents (L. latus and L. cardui) have now been in the eld for over six years. Their behaviour con rms the test outcomes. Adults have very occasionally been found on other species of introduced carduine thistle near Onopordum infestations, but there is no evidence of being able to develop on species other than the target weeds. With regard to the broader issue of the methodology of host-speci city testing, this study is instructive as the results of testing eight species, from diverse taxa and occupying diverent feeding niches, can be compared. Most publishe d data on the host-speci city of weed biologica l control agents concern only one or occasionally two species (108 and 11 papers, respectively, published over the last 10 years based on a search of the ISI Web of Science database) and hence tend to be mainly descriptive. The results here demonstrate the value of a exible, rather than a formulaic approach to host-speci city testing. The latter (e.g. Wan & Harris, 1997; Olckers, 2000), in which prescribed tests are applied to all stages of the agent for all test plant species, can greatly increase the cost of testing without improving the ultimate assessment of risk. In the present study, only one method of testing was needed for four of the eight agents, two for a second species and a third for the remaining three species. As Sheppard (1999) has pointed out, agent biology should dictate the insect life-stages to be tested. This study also demonstrates how the behaviour of diverent insect species is diverentially in uenced by removing both host choice and mobility. In some species, host range was unavected, while other showed some reduced speci city and others laid indiscriminately on non-host plants, compared to their behaviour in open- eld trials. These trials also demonstrated that behaviour constrains the physiological host-range, as three of the insects, L. latus, E. amoena and T. sulphurea were found to be capable of developing on some species closely-related to carduine thistles (see also van Klinken & Heard, 2000). There is also a suggestion that ovipositio n behaviour may be more avected by containment and restriction of choice in insects where adults have a similar feeding habit to the larvae (e.g. both feed on leaf-tissue), than where this is diverent (e.g. adults are nectar-feeders while larvae feed on plant tissue), and in those that have mobile larval stages than those that have immobile, endophagou s larvae (where `mistakes may have more dire consequences). A wider analysis is needed though to see if this is a general trend. Notwithstanding, the work here demonstrates the value of initially using no-choice cage tests. While these tests can produce false positive results, due to the evect of con nement on insect behaviour, such errors are conservative, which means that negative results are extremely robust (Hill, 1999). As indicated above, no further tests were required for half the species tested following the initial no-choice test. In the present study, where positive results occurred in the initial series of no-choice tests, clari cation was obtained either by re-testing in a choice situation and/or by the results of testing host-range of other life-stages. Those agents tested showed a reduced host-range when a choice between target and non-target plants was permitted, while larval development tests demonstrated that any risk suggested by adult ovipositio n tests was inconsequential. The results of preliminary open- eld choice tests were also useful in helping to interpret the data from more behaviourally restrictive quarantine tests. Finally, with regard to the issue of the selection of test plants, this study demonstrates that a test list can be safely based solely on phylogenetic grounds, without the need to test `safeguard species that are important for economic or conservation reasons but are more distantly related to the target (as recommended by Wapshere (1974) and carried out in most cases of host-testing publishe d to date). An improved knowledge of plant phylogeny and the evolution of host utilization should enable a greater reliance on science rather than fear

15 HOST SPECIFICITY OF CANDIDATES AGAINST THISTLES 161 in the risk assessment of biologica l control agents. While the current testing procedure is known as the `centrifugal phylogenetic method (Wapshere, 1974), in practice it uses taxonomic circumscription (the delineation of organisms into named taxa such as families, tribes and genera) rather than phylogeny sensu stricto (the evolutionar y relationship between organisms) to generate test plant lists. The latter provides a better model for viewing the relationships of test plant species to the target weed. For example, in the current study there are ve degrees of separation between clades of test plants and Onopordum spp., including two clades within related genera and three clades within the related tribes (Figure 1). The tests conducted here revealed that non-target evects decreased with increasing degree of separation from Onopordum spp., con rming the utility of this approach. The phylogeneti c view not only provides ner resolution of relationships, it also shows those that are at a similar level, not all of which might need to be tested. Thus, in the present study, perhaps even fewer test species would have been necessary to produce a result with the same degree of con dence, by only testing two or three species within the more distant clades, rather than testing a representative of each tribal circumscription. Overall, the present study leads to a number of recommendations for host-speci city testing of arthropods used for weed biologica l control in general, con rming some practices and suggesting others: Where possible, preliminary agent testing should be done in the area of origin against key plant species (i.e. at least those with only one degree of phylogenetic separation) to determine whether the cost and evort of formalized testing is worthwhile. If feasible, open- eld experiments in the area of origin are preferable, as they can aid interpretation of more formal quarantine testing. Test plant lists to be approved for formal host-speci city testing should be based strictly on phylogenetic grounds when this information is available. The life-stage(s) to be used for systematic testing of an approve d test plant list should be determined from the knowledge of agent biology and behaviour. Formalized testing should initially be done using no-choice tests to promote the most extreme behavioural response of the agent. Ambiguous responses should then be checked using choice experiments, aided by data from open- eld trials where this is available. ACKNOWLEDGEMENTS This work has been supported by Australian Wool Innovatio n and Meat and Livestock Australia. Thanks are due to Thierry Thomann and Janine Vitou for technical support in Montpellier. We would also like to thank Tim Heard for comments on an earlier draft of this manuscript. REFERENCES Amaral Franco, J. Do (1976) Onopordum, in Flora Europaea, Vol. 4 (Tutin, T.G., Ed.). Cambridge University Press, Cambridge, pp Angiosperm Phylogeny Group (1998) An ordinal classi cation of the families of owering plants. Annals of the Missouri Botanic Gardens 85, Bremer, K. (1996) Major clades and grades of the Asteraceae, in Compositae: Systematics. Proceedings of the International Compositae Conference, Kew, 1994 (Hinds, D.J.N. & Beentje, H.J., Eds). Royal Botanic Gardens, Kew, Vol. 1, 1-7. Briese, D.T. (1989) Host-speci city and virus-vector potential of Aphis chloris Koch (Hemiptera: Aphididae), a biological control agent for St John s wort in Australia. Entomophaga 34, Briese, D.T. (1999) Open eld speci city tests: Is `natural good enough for risk assessment? In Host SpeciWcity Testing in Australasia: Towards Improved Assays for Biological Control (Withers, T.M., Barton Browne, L. & Stanley, J., Eds). CRC Tropical Pest Management, Brisbane, Australia, pp

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