A retrospective evaluation of the host range of four Aphidius species introduced to New Zealand for the biological control of pest aphids

Biological Control 67 (2013) 275–283

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A retrospective evaluation of the host range of four Aphidius species introduced to New Zealand for the biological control of pest aphids P.J. Cameron a,⇑, R.L. Hill b, D.A.J. Teulon c, M.A.W. Stufkens c, P.G. Connolly d, G.P. Walker d a

20 Westminster Road, Mt Eden, Auckland 1024, New Zealand Richard Hill and Associates, Private Bag 4704, Christchurch, New Zealand c New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch, New Zealand d New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The host range of four Aphidius spp.

introduced for biological control was reassessed.  No-choice and choice tests were compared with field literature records.  Aphidius eadyi and A. sonchi were confirmed as largely specific to target (control) aphids.  A. ervi and A. rhopalosiphi parasitized some native aphids at a low rate.  Tests on newly discovered native aphids would be required to change historic regulatory decisions.

a r t i c l e

i n f o

Article history: Received 8 April 2013 Accepted 13 August 2013 Available online 24 August 2013 Keywords: Aphid parasitoids Classical biological control Host tests Regulatory decisions

a b s t r a c t Four species of Aphidius (Hymenoptera: Braconidae) were deliberately established in New Zealand in the period 1977–1994 for the biological control of pest aphids. Biological control practice and its regulation evolved over this period, so that whereas the evidence required for the 1977 introductions was based on literature records, by 1994 additional experimental or observational information was required. This paper describes the use of no-choice and choice tests conducted in 1996 and 1997 to retrospectively evaluate parasitoid host ranges and it considers if this information would alter the original regulatory decisions. The test species included several native aphids that were unknown at the time of the original parasitoid introductions. Consistent with literature records, the experiments confirmed that Aphidius eadyi Stary, Gonzalez and Hall was specific to its target host, and Aphidius sonchi Marshall was largely specific. Aphidius ervi Haliday and Aphidius rhopalosiphi De Stephani-Perez parasitized several test aphid species including cosmopolitan pest species already recorded in the literature, and some native test species. The patterns of mummification of individual test aphids varied greatly. Test aphids in the subfamilies Saltusaphidinae, Calaphidinae and Neophyllaphidinae appeared not to be at risk at all. The native Aphidini species Paradoxaphis plagianthi Eastop appeared to be more susceptible to attack by both A. ervi and A. rhopalosiphi than were P. aristoteliae Sunde or Aphis spp., suggesting that further investigations of P. plagianthi would be a priority if the introduction of these parasitoids were reconsidered now. Because P. plagianthi was not known when A. ervi and A. rhopalosiphi were introduced, prediction of their subsequent host range was limited by knowledge of the aphid fauna at that time. Therefore, reassessments of any decisions to release particular parasitoids would not be altered significantly by tests using knowledge available at the time of their introduction. However, if introduction of these parasitoids were to be considered today there would need to be a greater emphasis on determining their impact on (the recently documented) native aphid species. Ó 2013 Elsevier Inc. All rights reserved.

⇑ Corresponding author. E-mail address: [email protected] (P.J. Cameron). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.08.011

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1. Introduction Four species of Aphidius (Hymenoptera: Braconidae) were deliberately established in New Zealand in the period 1977–1994 for the biological control of pest aphids (Cameron et al., 1989; Teulon et al., 2009). In each case, permission to release the control agent in New Zealand followed a regulatory judgment that the potential benefit of introduction outweighed any potential adverse environmental or economic effects. However, biological control practice and its regulation evolved over this period, so that whereas the evidence required for the 1977 introductions was based on literature records, by 1994 additional experimental or observational information was required. Over the period of introductions described in this paper, decisions and criteria for release were initially made under three pieces of New Zealand legislation; the Plants Act 1970; the Animals Amendment Act 1990; while current decisions are made under the Hazardous Substances and New Organisms (HSNO) Act 1996. Under the Plants Act 1970, although environmental issues were not specified, information on host range was required for a permit to import, and introductions were to be in the public interest (Longworth, 1987). The Animals Amendment Act increased the emphasis on environmental effects and requirements for consultation, and the HSNO Act 1996 requires the regulator to decline applications if the organism is likely to cause any significant displacement of a native species in its natural habitat. This new standard significantly increased the formal requirement for knowledge of the host range of parasitoids and was associated with increasing concern for possible non-target impacts from biological control agents including aphid parasitoids (Stufkens and Farrell, 1994). This paper asks whether the original decisions to introduce aphid parasitoids would have been made had the more demanding standards for demonstrating environmental safety been applied. Factors that influence host selection by parasitoids of arthropods are varied and complex and, increasingly, experimental estimation of host ranges is required for evaluation of introductions for biological control (Van Driesche and Hoddle, 1997). Phylogenetic approaches have been less successful for entomophagous biological control agents than for weed biological control agents (Van Driesche, 2004). This difference may stem from the tritrophic effects of host plants, previous aphid hosts, and parasitoid genotype on parasitoid host range. These factors have been extensively investigated in aphid parasitoids, particularly Aphidius ervi Haliday and A. rhopalosiphi De Stephani-Perez (Cameron et al., 1984; Powell and Wright, 1988, 1991) and more recently have been used to characterize habitat specialism (Stilmant et al., 2008) in these species. Our experiments were based on assessments of physiological suitability using no-choice experiments and host preference using choice experiments. Habitat or host location and host acceptance were not directly investigated. In 1996 and 1997 a series of laboratory experiments were conducted to compare and contrast the physiological host ranges of the four Aphidius species that had been introduced to New Zealand. The tests were performed on a range of native and introduced aphid species, and included several native species that were rare, undescribed, or unknown at the time of the original parasitoid introductions. New information on the native aphid species was generated partly by the need to locate and describe species in order to evaluate parasitoid host ranges in the experiments outlined here. Since 1977, when parasitoid introductions commenced, an additional 10 putative native aphid species have been located, rediscovered or described (Macfarlane et al., 2010; Teulon et al., 2013). Test species were selected from aphid subfamilies and tribes – using classifications based on von Dohlen et al. (2006) – including native and non-native species in an attempt to identify

phylogenetic preferences that have been described in the literature. Here the original host range data from the experiments are used to investigate potential field host ranges and reassess the decisions to introduce the parasitoid species. Several factors including the availability of new indigenous aphids as additional test species, new test techniques and new regulatory criteria would be expected to contribute to any differences between actual and retrospective decisions. The usual post-release standard for evaluating any form of host testing, including the laboratory host tests conducted in this study, is the realized host range determined from field studies following the establishment of biological control agents. For this study data on field host range from ongoing collections summarized by J.A. Farrell, P.J. Cameron and M.A. Stufkens (unpublished), together with published records from overseas surveys, were used to evaluate two aspects of biological control practice: i How well do laboratory experiments conducted prior to release predict subsequent field host range? ii When faced with limited laboratory-based data, do regulatory authorities make appropriate decisions about whether to release an agent or not? The laboratory experiments are reported here and compared with information known about the parasitoids at the time of introduction and discussed in relation to subsequent information on their host ranges. 1.1. Importation and release Five species of Aphidius have been introduced to New Zealand in three classical biological control programs over a period of 15 years, and four species have established (Teulon et al., 2009). Aphidius eadyi Stary, Gonzalez and Hall was imported in 1977 as a biological control agent for A. pisum (Harris) (Cameron et al., 1979). Originally sourced from Morocco, the founding culture was obtained via the University of California lucerne aphid program (Gonzalez et al., 1978). This parasitoid has been implicated in the decline of pea aphid populations (Cameron and Walker, 1989; Cameron et al., 1981). Aphidius smithi Sharma & Subba Rao was obtained from the same source but did not establish in New Zealand. A. ervi was imported from 1977 to 1981 as a biological control agent for Acyrthosiphon kondoi Shinji, bluegreen lucerne aphid, and A. pisum. Cultures obtained from University of California, Riverside in 1977 were originally sourced from Japan, Israel, Greece, Tashkent and Belgium (Cameron et al., 1979). All were released at some time between 1977 and 1981. Material sourced from Tasmania (1979–81) and Rothamsted, UK (1981) was also released (Cameron and Walker, 1989). It was later found that this species, or a species closely related to A. ervi (Carver, 2000), was present in New Zealand in 1963, decades before it was deliberately introduced (Teulon et al., 2009). A. rhopalosiphi De Stephani-Perez was imported from southern England and western France in 1985 as a biological control agent for control of Metopolophium dirhodum (Walker), rose-grain aphid (Stufkens and Farrell, 1987) and has established in all release areas and spread rapidly. Aphidius sonchi Marshall was imported from south-east Australia in 1992 as a biological control agent for Hyperomyzus lactucae (L.), sowthistle aphid (Stufkens and Farrell, 1995). It established but was also recovered in some areas where it had not been released, suggesting it was present before releases took place (Stufkens and Farrell, 1995).

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1.2. Recorded host range at introduction At the time of their introduction the Aphidius species used in these biological control programs each demonstrated a different range of reported host specificity in the field, from oligophagy in A. ervi to specificity in A. eadyi. Although some of these records are likely to emphasize pest species, they allow comparisons of the frequency of hosts among aphid subfamilies, tribes and subtribes. When described as a species, A. eadyi was considered to be restricted to host aphids in the genus Acyrthosiphon (tribe Macrosiphini) (Stary et al., 1980). The species was first recognized (Gonzalez et al., 1978) during the Californian lucerne aphid biological control program and was shown to develop on A. pisum but not on A. kondoi, nor Rhopalosiphum spp. (tribe Aphidini) (Stary, 1978; Stary et al., 1980). On introduction to New Zealand, A. eadyi was considered to be a specific parasitoid of A. pisum. Host records for A. ervi varied considerably between Europe and North America. In early North American literature this parasitoid was considered to be specific to Acyrthosiphon spp. (Mackauer and Finlayson, 1967). European literature considered A. ervi to be an oligophagous parasitoid mainly of the Macrosiphini, with some records from the Aphidini, subtribe Rhopalosiphina (Stary, 1962, 1974, 1976). In this literature, records of A. ervi from the subtribe Aphidina are rare, and the species are not reported. Stary (1974) listing of Aphis as a host (in his Table 1) was noted as Aphis sp. and Stary (1976) lists ‘‘Aphis sp – s. France’’ as a host. Marsh (1977) does not give his source for host information but refers to Stary (1974) as above. Therefore, in the 1970s, there may have been only one original source of information for Aphis as a host, with no record of the species. At the time of its introduction to New Zealand A. ervi was considered to be a parasitoid of the Macrosiphini and Rhopalosiphina, but not the Aphidina (P.J. Cameron, unpublished). In Europe, A. rhopalosiphi was noted as the most common parasitoid of the macrosiphines M. dirhodum (Rabasse and Dedryver, 1983), Sitobion avenae (F.) (Stary, 1978) and Sitobion fragariae (Walker) (Pungerl, 1984). It was also recorded from Rhopalosiphum padi (Aphidini: Rhopalosiphina) (Stary, 1978).

Table 1 Subfamily or tribe of test aphids, ⁄ = native species. Subfamily/Tribe and species Aphidini ⁄Aphis coprosmae Laing ex Tillyard Aphis epilobii Kaltenbach Aphis gossypii Glover ⁄Paradoxaphis aristoteliae Sunde ⁄Paradoxaphis plagianthi Eastop Rhopalosiphum padi (L.) Macrosiphini Acyrthosiphon kondoi Shinji Acyrthosiphon pisum (Harris) Brachycaudus rumexicolens (Patch) Brevicoryne brassicae (L.) Hyperomyzus lactucae (L.) Lipaphis pseudobrassicae (Davis) Metopolophium dirhodum (Walker) Myzus persicae (Sulzer) Sitobion fragariae (Walker) Uroleucon sonchi Marshall Calaphidinae Myzocallis coryli Goetze Neophyllaphidinae ⁄Neophyllaphis totarae Cottier Saltusaphidinae Thripsaphis foxtonensis Cottier

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Field records prior to the introduction of A. sonchi showed that it was restricted to Hyperomyzus species (Mackauer and Stary, 1967; Stary, 1976) and in host range tests in New Zealand, parasitism was ‘‘zero or negligible’’ (Stufkens and Farrell, 1994) in seven species of Aphis and Drepanosiphinae related to native aphids. Laboratory parasitism of Uroleucon sonchi was contradicted by lack of parasitism of this species when associated with H. lactucae in the field (Aeschlimann and Vitou, 1985). 1.3. Experimental evaluations There was no experimental evaluation of the potential impacts of A. eadyi, or A. ervi on non-target aphid species prior to release in New Zealand (R.L. Hill, P.J. Cameron unpublished). Experiments to determine the host range of A. rhopalosiphi conducted prior to release were restricted to exotic aphid species (Teulon et al., 2009). One of the five native aphid species available at the time of the introduction of A. sonchi was tested before this species was introduced (Stufkens and Farrell, 1994). Since that time, the number of native aphid species known in New Zealand has grown to 15 or more (Teulon et al., 2009, 2013). The susceptibility of selected test aphids to attack by these parasitoids in the presence and absence of the target host was examined retrospectively in the new series of laboratory experiments performed from 1996 to 1997. The purpose of these experiments was to examine the robustness of the risk analysis that led to the decision to release, and to look for patterns of attack that might help predict risk to species that were not tested. 2. Materials and methods 2.1. Insect material The test aphid species were chosen to represent a range of the subfamilies, tribes and subtribes present in New Zealand, as listed in Macfarlane et al. (2010). They included cosmopolitan and native species as well as pests and non-pests (Table 1). Notably, A. ervi and A. rhopalosiphi were introduced as species with several pest aphid targets, and several test species were also known hosts. Additionally, the choice of test aphid species was determined by availability of sometimes rare species, and therefore varied among experiments (Fig. 1). It aimed to include native species and, in some instances, test species included aphids that were not known at the time of parasitoid introduction. Control aphids were chosen from the major species targeted by each introduction and are listed in Table 2. Parasitoid cultures originated mainly from the Canterbury region, but were also sourced from Manawatu and Auckland. The experiments were performed from July 1996 to July 1997 and new field collections were undertaken to refresh the cultures during the course of the experiments to reduce the development of laboratory strains. 2.2. Experimental procedures Winged aphids were placed on plants and allowed to produce young for 1–2 days to provide a standard source of late first to second instar control and test nymphs. In simple no-choice tests, the target aphid (control) and test aphids were exposed to parasitoids in separate ventilated boxes measuring 200  120  70 mm placed on end. Two 10 cm sprigs of the preferred host plant of the target aphid species were placed in water in each of two 25 mm diameter tubes in one box, and 50 of these aphids were added to each shoot as controls. In each box, two sprigs of the normal host plant of the test aphid were arranged, and 50 test aphids were added to each sprig.

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Fig. 1. Percent parasitism (±2 SE) (measured as rate of mummification) of test aphids compared with target (control) aphids (⁄ = native species) in choice and no-choice tests. All aphids are listed and those not tested for a particular parasitoid are left blank. The control species for Aphidius eadyi is Acyrthosiphon kondoi, for Aphidius ervi is Acyrthosiphon kondoi, for A. rhopalosiphi is Metopolophium dirhodum, and for A. sonchi is Hyperomyzus lactucae. Letters indicate aphid subfamilies or tribes: A = Aphidini, M = Macrosiphini, C = Calaphidinae, N = Neophyllaphidinae, S = Saltusaphidinae. NS denotes no significant difference between test and control parasitism at the 5% error level.

In choice tests, two aphid species were presented to parasitoids in a single box – the target aphid of the parasitoid under evaluation and a test aphid species. A 10 cm sprig of the preferred host plant of the control aphid was arranged in one tube, and that of the test aphid in the other. Approximately 50 test and 50 control aphids were transferred to the appropriate host plant. One male and two female parasitoids were transferred to each box. These were selected randomly from 2-day-old adults that were emerged into small containers with no aphids or plant material, and allowed to mate and feed on a parasitic Hymenoptera diet (20% sugar, 20% honey, 1% agar) formed into droplets. Tests were conducted at 20 °C and 65–70% RH with a light: day cycle of 16:8 h. After 24 h the parasitoids were removed and the cuttings were maintained for 8–11 days for mummification of parasitized

Table 2 Target or control aphid species for each parasitoid. Parasitoid

Target (control) aphid

Aphidius Aphidius Aphidius Aphidius

Acyrthosiphon pisum (Harris) Acyrthosiphon kondoi Shinji Metopolophium dirhodum (Walker) Hyperomyzus lactucae (L.)

eadyi Stary, Gonzalez and Hall ervi Haliday rhopalosiphi De Stephani-Perez sonchi Marshall

aphids to be completed. Mummies were counted together with the remaining aphids and the rates of parasitism were calculated as mummies per aphid. Each test and control was replicated three times.

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2.3. Analysis The number of aphids in which mummification was observed was analyzed in a generalized linear model using R (R Development Core Team, 2012). A quasi-poisson model was used to make adjustment for the high degree of under-dispersion or sometimes over-dispersion. The model incorporated the number of aphids available to a prospective parasitoid and was used to predict the percentage of mummified aphids for each combination of parasitoid and aphid for choice and no-choice studies. For each model we calculated the probability of obtaining test and control results as different (P < 0.05) as those observed under the null hypothesis of no difference in the susceptibility of the aphids to the parasitoid. A quasi-binomial model analyzing the data as proportions was also tried. It gave almost identical results. In addition to the specific tests, plots of the mean plus or minus two standard errors were used to indicate approximate significant differences at P = 0.05. In the cases of zero mummification, the p-value under the null hypothesis becomes meaningless. For specific comparisons of no-choice tests with A. ervi, assuming the preferences of the parasitoid did not change over the trials, the quasi-poisson model was adjusted to estimate if mummification of Paradoxaphis plagianthi Eastop was similar to that of selected species. To allow for the fact that multiple comparisons were being made simultaneously, the multcomp (Hothorn et al., 2008) R package was used with Dunnett’s contrasts (Dunnett, 1955) which effectively reduces the required level of alpha necessary to reject the null hypothesis. A multi-panel Lattice plot (Sarkar, 2008) was used (Fig. 1) to show the substantial separation between the degree of mummification of test and control aphid species, and a small degree of difference between the choice and the no-choice observations. The horizontal lines slightly above or below the line of the plotting characters represent two standard errors above and below the predicted mean (on the log scale). The letters NS against the left edge of a panel indicate that the difference between the control and test is not significant at the 5% error level, and error bars are absent where they are not calculable. The capital letter on the right edge groups aphid species according to subfamily or tribe. Simple rankings were used to compare susceptibility of tribes, with zero where no species were parasitized. Numerical values for percent parasitism (measured as rates of mummification) in Fig. 1 are provided in Appendix A (Supplementary data).

3. Results These experiments demonstrated significant differences among the physiological host ranges of the parasitoid species and provided a basis for identifying which host species may be relatively at risk in particular parasitoid-host combinations (Fig. 1). The variance in the rate of parasitism (measured as mummification) within host species (indicated as 2 SE) was low enough to distinguish between control and test parasitism for all but five test species that are recognized as pest species themselves, and one native species. Test parasitism was not significantly different from controls where marked ‘‘NS’’. This occurred when there were high or variable rates of parasitism on some pest species; viz. A. pisum, Myzus persicae (Sulzer), S. fragariae, U. sonchi, and Aphis gossypii Glover, that are recognized as host species, and for the native Paradoxaphis aristoteliae Sunde. Lack of parasitism in several parasitoid-aphid combinations demonstrated that those test aphids were not physiological hosts, but the extent to which low parasitism is associated with non-host status still relies on external validation from field and literature records. The presence of test species (in choice experiments) or their absence (no-choice experiments) as alternative hosts had little

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influence on the average performance of parasitoids on target (control) hosts (Fig. 1). However, variability in parasitism of target hosts was greater in choice tests with A. sonchi suggesting that host preference cues had some effect when target species were also present. Average parasitism rates for test species were similar in choice and no-choice tests, and for both A. ervi and A. rhopalosiphi it was equally likely that parasitism in one test would exceed the other. However, differences between individual test aphid species (Fig. 1 and below) were most important for assessing the specificity of parasitoids. Data from no-choice tests were emphasized unless indicated. 3.1. A. eadyi Of the 17 test species presented to A. eadyi in these tests, four were native species. Average parasitism was similar in test aphids with averages of 0.2% in no-choice tests and 0.3% in choice tests (Fig. 1) and showed significant (or non-testable for zeroes) differences between all test species and the control aphid, A. pisum. Zero parasitism was recorded for most test species. The remaining aphids were parasitized at low rates of 0.5–0.8% for those in the tribe Aphidini and 0.4–2.8% for those in the Macrosiphini. Only one native species (Aphis coprosmae Laing ex Tillyard) was parasitized, producing a single mummy. The remaining five test aphids that produced mummies were parasitized at levels significantly (P < 0.01) below the parasitism recorded for the control, and all but one (with two mummies in one replicate of H. lactucae) of these test replicates produced only a single mummy. 3.2. A. ervi Sixteen test species were presented to A. ervi including three native species, using A. kondoi as the control. Mummies were formed in all but two test species and this was at a very variable rate. Average parasitism of test aphids was 10.6% in no-choice tests and 7.8% in choice tests (Fig. 1). In no-choice tests mean parasitism of two known hosts, A. pisum and M. persicae, was above 30% but was below 20% in the remaining species. Mean parasitism of A. pisum was not significantly different from the control A. kondoi. When host species were ranked for susceptibility to parasitism, no parasitism was recorded in the subfamilies Calaphidinae and Neophyllaphidinae, whereas the mean rank was 8.4 for species belonging to the tribe Aphidini, and 9.8 for the Macrosiphini. These ranks suggest that subfamily but not tribal differences are a factor in susceptibility to this parasitoid. In the Macrosiphini the highest rates of parasitism occurred on known hosts A. pisum (75%) and M. persicae (36%), neither of which were significantly different from controls (P > 0.05), followed by S. fragariae (22%) and B. rumexicolens (Patch) (15%) which were significantly lower than controls (P < 0.05). The highest rates of parasitism in the Aphidini was P. plagianthi with approximately 14% in both tests, but significantly lower than controls (P < 0.05). Specific contrasts (Dunnett, 1955) with P. plagianthi showed that parasitism of this species was significantly less (P = 0.027) than M. persicae (36%), and similar (P = 0.993) to that of S. fragariae (12%), and could not be separated from L. pseudobrassicae (6.3%, P = 0.253), Aphis epilobii Kaltenbach (4.4%, P = 0.126) and U. sonchi (1.8%, P = 0.059). Parasitism of all other Aphidini was below 2% and was significantly lower than A. kondoi controls. 3.3. A. rhopalosiphi Fourteen test species were presented to A. rhopalosiphi including four natives. Average parasitism of test aphids was similar in no-choice tests (13.6%) and in choice tests (11.9%) (Fig. 1). The known hosts M. persicae, R. padi and S. fragariae recorded above

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30% parasitism. Parasitism of S. fragariae was not significantly different from the control species M. dirhodum, but this was associated with highly variable results. The remaining species were arbitrarily classified into two groups in no-choice tests, with five species between 5% and 20% parasitism, and six species below 3%. Apart from the two known hosts, parasitized aphids belonging to the tribe Aphidini and Macrosiphini occurred similarly in these groups, and parasitism of both native species was below 3%, but in choice tests P. plagianthi recorded 10.4% parasitism. No parasitism was recorded in the subfamilies Calaphidinae and Neophyllaphidinae. 3.4. A. sonchi Eighteen test species including all five natives were presented to A. sonchi. Average parasitism of test aphids was 1.0% in nochoice tests and 1.9% in choice tests. Associated with the greater variability in the choice test controls, parasitism of U. sonchi and P. aristoteliae was not significantly different from the control (Fig. 1). Maximum parasitism in no-choice tests was 7.5% on the recorded host U. sonchi, with 3.9% of P. plagianthi, 3.5% of A. epilobii and no parasitism on 12 species (Fig. 1). No parasitism was recorded in the subfamilies Saltusaphidinae, Calaphidinae and Neophyllaphidinae. 3.5. Comparisons with literature Comparison of field host records from the literature with our testing information for each parasitoid species (below) provided an evaluation of their combined value in predicting their host range as currently known from the latest overseas field surveys and New Zealand records (J.A. Farrell, P.J. Cameron and M.A. Stufkens, unpublished). 3.5.1. A. eadyi Following its introduction to New Zealand as a specific parasitoid, A. eadyi was confirmed as specific to A. pisum in laboratory tests and was absent from A. kondoi in the field (Cameron and Walker, 1989). The host testing in the current study retrospectively confirmed this specificity. Low rates of parasitism including 0.5% parasitism of A. coprosmae suggest that confusion of host location cues in confinement (Sands, 1993) lead A. eadyi to oviposit rarely in some non-hosts. Overseas surveys (Kavallieratos et al., 2004; Stary and Havelka, 2008; Tomanovic et al., 2003) and New Zealand records (J.A. Farrell, P.J. Cameron and M.A. Stufkens, unpublished) have since confirmed the absence in the field of A. eadyi from Macrosiphini other than A. pisum and its absence from aphids in the Aphidini. 3.5.2. A. ervi At the time of its introduction to New Zealand, field host records from the world literature suggested that A. ervi was a parasitoid of the Macrosiphini and the subtribe Rhopalosiphina but not the subtribe Aphidina (including Aphis spp.). In the host tests reported here, A. ervi was reared from three additional Macrosiphini not noted in this literature, viz. Brevicoryne brassicae (L.), Lipaphis pseudobrassicae (Davis) and U. sonchi (L.). The continuing absence of these three hosts from more recent field surveys (Kavallieratos et al., 2004; Kos et al., 2009; Stary and Havelka, 2008; Tomanovic et al., 2003) suggests that the levels of laboratory parasitism that we recorded for B. brassicae, L. pseudobrassicae, and U. sonchi (0.7–6.3%) resulted from the confined testing regime, particularly since such low levels are consistent with test species that have not been confirmed as field hosts (Barratt et al., 1997). As none of these Macrosiphini are valued non-targets, their parasitism is not of environmental concern in New Zealand.

Host tests on the Aphidina demonstrated some physiological compatibility of A. ervi with four species including two natives, namely P. aristoteliae (2.5% parasitism) and P. plagianthi (14.3%). This level of parasitism placed P. plagianthi as less susceptible than the known host M. persicae, similar to another known host S. fragariae, and similar to a group of aphids (including L. pseudobrassicae, A. epilobii and U. sonchi), which suffered low levels of parasitism. Assuming that the A. ervi culture did not vary over time, these results place the susceptibility of P. plagianthi in the same group as the susceptible host species S. fragariae, and the non-hosts L. pseudobrassicae, A. epilobii and U. sonchi. Comparisons with field host records for putative A. ervi in New Zealand are unclear because of difficulties with parasitoid identification. In separate field collections, Aphidius spp. (not necessarily A. ervi) have been reared from three native Aphis spp. (Teulon et al., 2009) but identification is awaiting confirmation and DNA analysis (see below). For example, in field collections of Aphis cottieri Carver a small number of parasitoids have been identified as conspecific or closely related to A. ervi (Carver, 2000). In contrast, no A. ervi were found from cosmopolitan Aphis spp. (J.A. Farrell, P.J. Cameron and M.A. Stufkens unpublished) although other Aphidius spp. were reared. Similarly, field surveys in Europe (Kavallieratos et al., 2004; Kos et al., 2009; Stary and Havelka, 2008; Tomanovic et al., 2003) have not detected A. ervi from Aphis spp. (Aphidina) nor from the Drepanosiphinae. However, Malina and Praslicka (2008) used Aphis pomi de Geer as a host in extensive rearing experiments with A. ervi, apparently confirming the laboratory but not the field host status of Aphis spp. Further research currently underway on this species includes comparisons of the DNA of A. ervi and ervi-like species (S.R. Bulman and G.P. Walker unpublished). Historically, earlier self-introduction of A. ervi to New Zealand or the existence of an ervi-like species is suggested by three specimens considered to be near A. ervi (M. Carver pers. comm. to J.A. Berry) collected from Aulacorthum solani (Kaltenbach) in 1963. These origins may be resolved by DNA analysis, such as that performed on North American A. ervi, which identified a putative biological control introduction as the native A. ervi pulcher (Baker), a divergent haplotype of A. ervi (Hufbauer et al., 2004). 3.5.3. A. rhopalosiphi Laboratory host tests showed a physiological range that is larger than the field host range revealed in the literature records. European records subsequent to the introduction of A. rhopalosiphi to New Zealand (Kavallieratos et al., 2004; Stary and Havelka, 2008; Tomanovic et al., 2003) mostly confirm the original restriction of this parasitoid to Metopolophium and Sitobion (Macrosiphini) and Rhopalosiphum (Aphidini: Rhopalosiphina), with no records from Aphis spp. (Aphidina). However, our testing recorded four laboratory hosts in the Aphidina, with three exceeding 10% parasitism in some tests, including 10.1% in the native P. plagianthi. In the Macrosiphini, three positive host-testing records over 8% parasitism (B. brassicae 11.8%, L. pseudobrassicae 15.6%, U. sonchi 8%) conflict with an absence of parasitism of these species in the literature records. The explanation for such levels of parasitism in tests on apparently non-host cosmopolitan aphids may again be the result of confined testing conditions. None of these Macrosiphini are valued non-targets. Interestingly, M. persicae is largely absent (Desneux et al., 2006) from overseas field records, but in New Zealand relatively high parasitism (41.9%) in the laboratory tests coincided with its presence on M. persicae in the field (Farrell and Stufkens, 1990). 3.5.4. A. sonchi Low rates of parasitism in experimental tests with both the Macrosiphini and Aphidini were mostly in agreement with the

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literature records. The exception was parasitism of U. sonchi in both sets of tests, confirming the laboratory test records of Stufkens and Farrell (1994). These results were contrary to field records (Aeschlimann and Vitou, 1985). The negative field observation was favored by Stufkens and Farrell (1995) to support claims for the specificity of A. sonchi to H. lactucae, and was accepted to gain permission to release this parasitoid. Subsequent surveys in Europe (Kavallieratos et al., 2004; Tomanovic et al., 2003) and New Zealand (J.A. Farrell, P.J. Cameron and M.A. Stufkens unpublished) have confirmed field specificity to Hyperomyzus spp.

4. Discussion Overall, the laboratory experiments indicate that A. eadyi was functionally specific to its target host with rare mummification of one or two aphids. Similarly low levels of parasitism have previously been associated (Barratt et al., 1997) with low realized host range of biological control agents in the field. A. sonchi was also largely specific, and although parasitism of P. plagianthi reached 3.9%, this level is also consistent with a low realized field host range. In contrast, in these laboratory tests, A. ervi and A. rhopalosiphi parasitized several test aphid species including cosmopolitan pest species already recorded in the literature and native species. The patterns of parasitism of individual test aphids varied greatly. Test aphids in the subfamilies Saltusaphidinae (Thripsaphis foxtonensis Cottier), Calaphidinae (Myzocallis coryli Goetze) and Neophyllaphidinae (Neophyllaphis totarae Cottier) appeared not to be at risk at all. The native Aphidini species P. plagianthi appeared to be more susceptible to attack by both parasitoids than was P. aristoteliae or Aphis spp. (Fig. 1). What makes one aphid species physiologically susceptible to attack and another immune was not made clear by this analysis, and this susceptibility or immunity varied with parasitoid species. Whereas the specificity of A. eadyi and A. sonchi was confirmed, A. ervi and A. rhopalosiphi exhibited wider physiological host ranges that were partly related to taxonomic distance from the target host. Factors influencing the host range of A. ervi and A. rhopalosiphi have been widely investigated. The sequence of presentation of host aphid species has been shown to influence subsequent host range in A. ervi (Cameron et al., 1984; Cameron and Walker, 1989), but testing for this effect requires extensive host-switching experiments not incorporated in the tests described in the present study. Although switching from Macrosiphini to Aphidini was demonstrated in our experiments, parasitism of Aphidini may have been reduced by previous laboratory selection for a Macrosiphini strain. Powell and Wright (1988) demonstrated that production of mummies by both A. ervi and A. rhopalosiphi in the laboratory is strongly influenced by the sequence of host aphids, and cautioned that laboratory selection influences experimental results. Attack rates by A. rhopalosiphi also increased on non-host aphids when wheat leaves were present (Powell and Wright, 1991; Braimah and van Emden, 1994). These studies lead to the conclusion that genotype determines the response of individual parasitoids to semiochemicals involved in host recognition (Powell and Wright, 1991). Thus, the test results obtained here reflect the host range of established field populations in New Zealand, which may vary from the original introductions. An additional host factor recently identified is the presence or absence of symbionts in host aphids that influence success of parasite development of A. ervi (Oliver et al., 2009). Given the difficulty of interpreting host range experiments, we agree with Van Driesche et al. (2003) that simple testing is only likely to confirm the specificity of parasitoids with narrow host ranges. Do these retrospective studies support the decisions to release these parasitoids in the first instance? It is generally accepted that

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closely confined experiments such as those described here are likely to overestimate the ecological host range of a parasitoid, because ecological filters and behaviors that mediate host-finding such as habitat complexity and long-distance detection in the field are compromised in such laboratory studies (Sands, 1993). Methods for investigating the role of host location in determining host range have been developed and utilized for testing parasitoids including Aphidius rosae Haliday (Kitt and Keller, 1998) and their use has been advocated for more effective specificity testing (Keller, 1999). Habitat associations deduced from extensive surveys (Kavallieratos et al., 2004; Stary and Havelka, 2008; Tomanovic et al., 2003) are also crucial for interpreting physiological testing to predict realized field host range or ecological host range. These field host ranges are invariably narrower than laboratory records, and suggest that the rates of parasitism measured in laboratory experiments represent a theoretical maximum field parasitism rate for the species tested. This conclusion is supported by the comparison of retrospective testing of Microctonus spp. in New Zealand with field records of parasitism (Barratt et al., 1997, 2010) showing that lower laboratory parasitism levels (in a range of 3–24%) were associated with <5% field parasitism of native species. To date, field records of the realized host range of A. ervi in New Zealand (J.A. Farrell, P.J. Cameron and M.A. Stufkens unpublished) support the overseas literature surveys indicating that laboratory parasitism is an overestimate of the host range, and therefore support the initial release decision. Current practice for interpreting host testing of parasitoids appears not always to provide a strong framework for generalizing from results such as those reported here. However, the tests do suggest that parasitism of non-target aphids by A. eadyi is likely to be rare or absent in the field, as was predicted by host records in 1977 and as borne out by recent surveys. Relatedness was a not a good predictor of susceptibility to parasitism for A. sonchi, but as no-choice test parasitism of natives was low it is considered unlikely that significant field parasitism would eventuate. In addition, the results add three further records to the list of native species immune from attack by A. sonchi, and results on other non-target species imply a high level of host specificity. On the basis of existing literature, these two parasitoid species are therefore (as predicted) unlikely to pose direct risk to non-target populations, and additional host testing would not have contributed substantially to decisions.A. ervi and A. rhopalosiphi parasitized several (but not all) of the non-target species presented in laboratory tests, but the parasitism rates were significantly lower than for the target and most other known hosts. As these rates were achieved in close containment, field rates of parasitism are likely to be lower. Of the native species tested, P. plagianthi appeared to be the most consistently susceptible, mostly to A. ervi, at moderate rates of about 14%. It seems unlikely that the rates of parasitism of native aphids observed in this experiment could be a key factor in population dynamics but, coupled with spatial effects and apparent competition, such rates could adversely affect populations locally. In particular the rarity of P. plagianthi (Kean and Stufkens, 2005) may increase the risk to this species locally, although existing parasitoids are thought by these authors not to be a key factor in the dynamics of this aphid. Quantitative studies of P. plagianthi (Kean, 2002) associated parasitism by Aphidius sp. with population crashes of this species, but the number of aphids killed was considered not enough to have caused the decline (Kean, 2002). Although broad generalizations could be made about the role of taxonomic isolation of aphids in mediating the host preferences of A. ervi and A. rhopalosiphi at the level of subfamily level, little can be concluded from these experiments about the risk that these parasitoids pose to native aphids that have not been tested. The host tests reported here do suggest that P. plagianthi should be a priority for further investigation. However, because this species

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was unknown when A. ervi and A. rhopalosiphi were introduced, testing was not an option then. Indeed, interest in such testing and concern for rare native species stimulated research on the aphid fauna and enabled the series of experiments that are reported here. Therefore, reassessments of any decisions to release particular parasitoids would not be altered significantly by tests using knowledge available at the time of their introduction. However, retrospective assessment of A. ervi and A. rhopalosiphi based on these host testing results and the current HSNO standards would be likely to require confirmatory host suitability tests native species, as well as additional tests including host location and host acceptability experiments to investigate ecological host ranges. Acknowledgments This project developed from research by the late John Farrell and Marlon Stufkens, and the experiments were designed and carried out by MAWS and DAJT. We thank Melanie Walker for technical help, and John Charles and Louise Malone for comment on a draft manuscript. This project was supported with funding from the New Zealand Government and Plant & Food Research as part of the Better Border Biosecurity (B3, www.b3nz.org) collaborative research programme. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocontrol.2013. 08.011. References Aeschlimann, J.P., Vitou, J., 1985. Aphids (Homoptera, Aphididae) and their natural enemies occurring on Sonchus spp. (Compositae) in the Mediterranean region. Acta Oecol. (Oecol. Appl.) 6, 69–76. Barratt, B.I.P., Evans, A.A., Ferguson, C.M., Barker, G.M., McNeill, M.R., Phillips, C.B., 1997. Laboratory nontarget host range of the introduced parasitoids Microctonus aethiopoides and M. hyperodae (Hymenoptera: Braconidae) compared with field parasitism in New Zealand. Environ. Entomol. 26, 694–702. Barratt, B.I.P., Howarth, F.G., Withers, T.M., Kean, J.M., Ridley, G.S., 2010. Progress in risk assessment for classical biological control. Biol. Control 52, 245–254. Braimah, H., van Emden, H.F., 1994. The role of the plant in host acceptance by the parasitoid Aphidius rhopalosiphi (Hymenoptera: Braconidae). Bull. Entomol. Res. 84, 303–306. Cameron, P.J., Walker, G.P., 1989. Release and establishment of Aphidius spp. (Hymenoptera: Aphidiidae), parasitoids of pea aphid and blue green aphid in New Zealand. N.Z. J. Agric. Res. 32, 281–290. Cameron, P.J., Thomas, W.P., Hill, R.L., 1979. Introduction of lucerne aphid parasites and a preliminary evaluation of the natural enemies of Acyrthosiphon spp. (Hemiptera: Aphididae) in New Zealand. In: Crosby, T.K., Pottinger, R.P. (Eds.). Proc. 2nd Austral. Conf. Grassland Invert. Ecol. Government Printer, Wellington, pp. 219–223. Cameron, P.J., Walker, G.P., Allan, D.J., 1981. Establishment and dispersal of the introduced parasite Aphidius eadyi (Hymenoptera: Aphidiidae) in the North Island of New Zealand, and its initial effect on pea aphid. N.Z. J. Zool. 8, 105–112. Cameron, P., Powell, W., Loxdale, H., 1984. Reservoirs for Aphidius ervi Haliday (Hymenoptera: Aphidiidae), a polyphagous parasitoid of cereal aphids (Hemiptera: Aphididae). Bull. Entomol. Res. 74, 647–656. Cameron, P.J., Hill, R.L., Bain, J., Thomas, W.P. (Eds.), 1989. A Review of Biological Control of Invertebrate Pests and Weeds in New Zealand 1874–1987. CAB International, Wallingford. Carver, M., 2000. A new indigenous species of Aphis Linnaeus (Hemiptera: Aphididae) on Muehlenbeckia (Polygonaceae) in New Zealand. N.Z. Entomol. 22, 3–7. Desneux, N., Rabasse, J.-M., Ballanger, Y., Kaiser, L., 2006. Parasitism of canola aphids in France in autumn. J. Pest. Sci. 79, 95–102. Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096–1121. Farrell, J.A., Stufkens, M.W., 1990. The impact of Aphidius rhopalosiphi (Hymenoptera: Aphidiidae) on populations of the rose grain aphid (Metopolophium dirhodum) (Hemiptera: Aphididae) on cereals in Canterbury, New Zealand. Bull. Entomol. Res. 80, 377–383. Gonzalez, D., White, W., Hall, J., Dickson, R.C., 1978. Geographical distribution of Aphidiidae (Hym.) imported to California for biological control of Acyrthosiphon kondoi and Acyrthosiphon pisum (Hom.: Aphididae). Entomophaga 23, 239–248.

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