SECTION 1 A description of the , biology, ecology and life cycle of the microhymenopteran wasp, Tachardiaephagus somervillei.

A. Taxonomy

Scientific name Tachardiaephagus somervillei (Mahdihassan, 1923) Common name None Relationships: Tachardiaephagus somervillei (Mahdihassan) is an encyrtid parasitoid:

Class: Insecta Order: Suborder: Superfamily: Chalcidoidea Family: Encyrtidae Subfamily: Encyrtinae

The Encyrtidae is one of the most important families of parasitic wasps () for the biological control of harmful , including a variety of scale insects infesting woody (Noyes and Hayat 1994, Noyes 2012). The Encyrtidae currently comprises 460 genera and 3735 in two subfamilies. The subfamily Encyrtinae includes 353 genera and 2920 species, while the Tetracneminae includes 107 genera and 815 species. Approximately half of all encyrtid species are associated with scale insects (: Coccoidea)(Noyes 2012). Encyrtids are generally endoparasitoids meaning that the parasitoid egg is laid directly inside the host’s body where the hatching larva completes development while feeding on host tissues, ultimately killing the host. Encyrtids mostly parasitize immature life stages (or, rarely, adults), but some species in one (Microterys) are egg predators (Noyes 2012).

B. Description

Tachardiaephagus somervillei was first described by Mahdihassan in 1923 as Lissencyrtus somervilli but Ferrière (1928) transferred L. somervilli Mahdihassan to Tachardiaephagus. He later downgraded this taxon from specific to subspecific rank as T. tachardiae somervilli (Ferrière 1935), based on the presence of intermediate forms and the lack of defining characters. This subspecies was accepted by Mahdihassan (1957). Varshney (1976) emended spelling of the subspecific rank to somervillei. In a recent reconsideration of the taxon, Hayat et al. (2010) elevated T. tachardiae var. somervillei to T. somervillei as a valid species. A detailed, formal taxonomic description of Tachardiaephagus somervillei does not exist. Although the specific name was erected by Mahdihassan (1923) he provided no accompanying morphological

1 description. In lieu of such a specific description, we first present a genus-level description based on the type species T. tachardiae (from Prinsloo 1977), and then note how T. somervillei differs from T. tachardiae.

Tachardiaephagus Ashmead Tachardiaephagus Ashmead, 1904: 303; Ferriere, 1928: 171; 1935: 396. Type-species Encyrtus tachardiae Howard, 1896. Lissencyrtus Cameron, 1913: 99; Ferrière, 1928: 171. Type-species Lissencyrtus troupi Cameron, 1913. The genus Tachardiaephagus was described from the type species, T. tachardiae (Howard) from South and Southeast Asia. For synonymy of the genus and species, descriptions and figures, consult Ferrière (1928, 1935), Prinsloo (1977), and Noyes (2012). The genus comprises seven species; four described from sub-Saharan Africa (T. absonus, T. gracilus, T. similis, and T. communis - Prinsloo 1977), and three from South and Southeast Asia (T. tachardiae, T. somervillei, and T. sarawakensis - Ferrière 1928, 1935, Hayat et al. 2010). All species are primary parasitoids known only from kerriid hosts ( - the of commerce, Paratachardina, and Tachardina species) (Prinsloo 1983). FEMALE. Primary parasitoids of . Medium-sized encyrtids, approximately 1.5-2.0 mm in length. Colour: head and body some shade of brown to black, the head and thoracic dorsum with or without faint to moderate metallic reflections; forewing hyaline or at most very faintly infuscated partly; hind wing hyaline. Head, in dorsal view (occiput perpendicular) with posterior margin moderately concave, the anterior margin convex, deeply notched by upper scrobal confluence (except in T. absonus); frontovertex united acutely or abruptly with occiput; frontovertex moderately broad, more or less than one-third head width at median ocellus; ocelli in an acute to obtuse-angled triangle; head, in frontal view, with toruli placed well above mouth margin, their upper limits approximately level with lower eye margins; scrobes strongly sulcate (except in T. absonus), long, their lateral margins sharply angled, confluent dorsally, impressed on face as an inverted 'V'. Antenna eleven-segmented; scape subcylindrical or at most moderately expanded ventrally, not less than three times as long as its greatest width; pedicel longer than basal funicle segment; funicle six-segmented, with all segments longer than wide or at most with distal one or two segments wider than long; club three-segmented, as long as or longer than the distal three funicle segments together, rounded apically, at most a little wider than funicle segment VI; maxillary palpi each with four segments, the labial with three; mandibles tridentate (sub-Saharan species) or with two teeth and a truncation (in T. tachardiae from South and Southeast Asia). Sculpture of head cellulate-reticulate, the cells relatively small, not raised, the frontovertex with fine setigerous punctations. Thorax moderately convex from side to side and anteroposteriorly, the mesoscutum plainly wider than long, the scutellum approximately as wide as long; mesoscutum without parapsidal sulci; mesoscutum with posterior margin overlapping mesal union of axillae partly or entirely, the latter not raised, separated medially by a narrow sulcus; sculpture of mesonotum cellulate-reticulate, the cells with very fine aciculations in some species; mesonotum moderately densely setose, the scutellum with one pair of suberect setae near apex. Legs not especially modified. Forewing

2 moderately broad, approximately 2.5 times as long as broad; venation well developed: submarginal vein slender; postmarginal vein usually longer than marginal, the former reaching to a level near apex of stigmal; wing disc evenly and rather densely setose from speculum to apex, the basal triangle never devoid of setae. Abdomen as long as or longer than thorax, the gaster usually heart-shaped, pointed or truncate at apex; cercal plates not strongly advanced towards base of gaster; gaster without paratergites; ovipositor varying in length, less than one-half to as long as gaster, as seen through the derm in cleared slide-mounted specimens; ovipositor protruding at most slightly caudally. MALE. Differing mainly from the female as follows: head and body generally black, with stronger metallic reflections (yellow variant in Oriental tachardiae). Head with frontovertex broader, approximately one-half head width at median ocellus; toruli placed higher on face, their lower margins about level with lower eye margins; ocelli in an obtuse-angled triangle. Antenna nine- segmented, the club not segmented, approximately as long as the distal two funicle segments together; flagellum with moderate to rather long, curved setae.

Tachardiaephagus somervillei (Mahdihassan 1923)(Fig. 1.1, Fig. 1.3A)

Lissencyrtus somervilli Mahdihassan, 1923: 71. Female. India, Karnataka. Tachardiaephagus somervilli (Mahdihassan): Ferrière, 1928: 71, taxonomy; Namkum (India) record. Tachardiaephagus tachardiae var. somervilli (Mahdihassan): Ferrière, 1935: 396, taxonomy; Malaysia record. Mahdihassan, 1957: 73-74. Tachardiaephagus tachardiae somervillei (Mahdihassan): Varshney, 1976: 61-62, emendation of specific name as it was based upon the name of Prof. Somerville.

No detailed species description is available for T. somervillei. However, Hayat et al. (2010) treat T. somervillei as a valid species as it differs from T. tachardiae not only in the yellow to orange-yellow colour of the head, but also in having F6 or F5 and F6 quadrate to slightly broader than long; and in having third valvula 0.73x mid-tibial spur. (In T. tachardiae: F5 and F6 longer than broad; and third valvula subequal in length to mid-tibial spur). Ferrière (1935) indicated that he found intermediate forms in two females from West Malaysia] (Rawang and Kuala Selangor Road) in which "the head has the vertex, the temples and cheeks dark green like T. tachardiae, and the face and inner margin of eyes reddish, as in somervilli". He, therefore, downgraded the species somervillei to a variety of tachardiae. However, Hayat et al. (2010) reinstated the species-level status for T. somervillei after examining specimens from Thailand and Malaysia and failing to find any specimens intermediate in head colour to T. somervillei and T. tachardiae.

C. Distribution, biology, behaviour and life cycle of the agent

Tachardiaephagus somervillei (Hymenoptera: Encyrtidae) is a primary parasitoid of kerriid lac scale species, including the yellow lac scale Tachardina aurantiaca (Hemiptera: Kerriidae), the target species for biological control on the Australian external territory of . It is an

3 endoparasitic species, meaning that it uses an ovipositor to insert eggs within the body of its female lac hosts. These eggs hatch and the larvae slowly consume the lac insect as it develops, finally pupating and emerging as winged adults, which results in the death of the host lac insect. Unlike the vast majority of encyrtids, T. somervillei attacks the mature stages of its hosts. The genus Tachardiaephagus has been known for almost a century due to the pest status of T. tachardiae in lac scale cultures in India and parts of Southeast Asia. Lac is produced from the lac scale, , and T. tachardiae and T. somervillei parasitize K. lacca, often causing economic injury (Narayanan 1962, Sharma 2006). Surprisingly, while the taxonomy of lac parasitoids as well as their control in lac production systems have been researched in India, detailed information about the life history and biology of T. tachardiae and T. somervillei is missing from the published literature. Some basic information on the life history of T. tachardiae is available, mostly from workers from the first half of the 20th century, but recent observations on T. somervillei extend and clarify this earlier research. Our observations in Peninsular Malaysia and Sarawak provide basic information on the distribution, biology, behavior and life cycle of T. somervillei in relation to its host, the yellow lac scale.

Distribution. Tachardiaephagus somervillei has a broad geographic range and has been recorded from India, Thailand, Peninsular Malaysia as well as Sarawak and Sabah in Borneo (Fig. 1.2). However, it has a highly restricted host range across this distribution, with all records from either Tachardina aurantiaca (southern Thailand, Peninsular Malaysia, Sarawak, and Sabah) or the lac insects of commerce Kerria (Bangkok, Thailand; India)(see Section 3 on host specificity testing for further details on host range).

Specimens of T. somervillei from West and East Malaysia are morphologically indistinguishable (M. Hayat, pers. comm. 2014), strongly suggesting that they are the same species. This conclusion was further supported by genetic barcoding of the mitochondrial cytochrome-c oxidase subunit 1 (COI) gene (600 base-pair sequence) of individuals from two locations in West Malaysia (Klang, n = 2; Selangor, n = 5) and one in East Malaysia (Kuching, n = 3). All sequences were identical, providing clear evidence of a single species with no indication of population differentiation across this considerable geographical range (Table 1.1).

Biology, behaviour and life cycle. Tachardiaephagus somervillei (Fig. 1.3A) attacks mature female hosts of Tachardina aurantiaca. Emerging females cut circular holes with smooth edges through the test, and oviposit soon afterwards (Fig. 1.3B, 3C). The importance of mating prior to oviposition is unknown. Search and oviposition behaviors in the presence of aggregations of its host Tachardina aurantiaca were observed for 28 female parasitoids under laboratory conditions. The female parasitoid walked relatively rapidly in the host aggregate, moving repeatedly over and between individual hosts rather than walking around them, even if there was a shorter path between them. Active walking in the aggregate may stop at any time; in the observation arenas the duration of activity was between approximately 1-8 minutes. Walking speed remained constant except either when the parasitoid is interested in honeydew or host acceptance and oviposition occurs. Host

4 acceptance is surprisingly quick with very little inspection by the parasitoid. It is possible that females inspected potential host individuals during prior short visits. When a host individual is accepted, the female assumes a typical oviposition posture with the head and thorax elevated and the abdomen pressed down (Fig. 1.3B). Narayanan (1962) claims that parasitoids of Kerria lacca, including Tachardiaephagus spp. oviposit through the anal pore of the host. This was not observed in oviposition on Tachardina aurantiaca. Instead, Tachardiaephagus somervillei oviposits in the upper part of the side of the host’s test. This may be an adaptive behavior: honeydew is excreted through the anal pore and could interfere with oviposition. Furthermore, tending T. aurantiaca collect honeydew and oviposition away from the anal pore may decrease the encounter rate between ants and T. somervillei. Oviposition itself is also relatively rapid, between 10-15 seconds. Once oviposition is completed, the parasitoid leaves the host immediately and if self-grooming takes place, it is usually done outside of the host aggregate. Without exception, inactivity was observed only after the parasitoid was outside of the host aggregate. The female may or may not groom after leaving the aggregate and enters an inactive state where it remains stationary. Inactivity ranged from just a few minutes to at least 30 minutes. Tachardiaephagus somervillei has multiple generations per year; closely related T. tachardiae has 9- 12 generations per year on the lac insect of commerce, Kerria lacca. Development time, from oviposition to adult emergence, was 23-26 days at 23-32 C. Although longevity under field conditions is not known, laboratory experiments indicate that water and nutrition are important; females (n = 14) provided with both water and 50% honey solution survived at least 33 days, but those (n = 14) denied access to both water and honey solution survived only 1-3 days. T. somervillei was observed utilizing honeydew from T. aurantiaca under field conditions, and it is probably an important food source that sustains adult parasitoids. Some parasitoids feed directly on their host by stabbing it with their ovipositor and feeding on the leaking hemolymph, but this has not been observed in T. somervillei. T. somervillei exhibits superparasitism, in which more than one offspring may emerge from a single female of Tachardina aurantiaca (Fig 1.3C). However, it is unknown whether this results from polyembryony (multiple embryos developing from a single egg), multiple eggs laid by the same female, or different females ovipositing multiple eggs. The number of successfully emerging progeny of T. somervillei is positively correlated with size of T. aurantiaca females (R2 = 0.47, n = 50, p < 0.001) (Fig. 1.4). Larger hosts are likely to provide more food resources, allowing more parasitoid larvae to complete development. This positive relationship between host size and production of T. somervillei progeny will be important in captive rearing, release site selection, and ensuring establishment during inoculative releases on Christmas Island. A variety of species, including the yellow crazy ant (Anoplolepis gracilipes) tend Tachardina aurantiaca in Peninsular Malaysia, Sarawak, and Sabah and collect honeydew (Fig. 1.3D,E; Table 1.2). Nevertheless, parasitism rates by Tachardiaephagus somervillei are high. Although ants are widely reported to interfere with search and oviposition by parasitoids (e.g., Way 1963), many parasitoids have sophisticated behavioural, chemical, and morphological adaptations that avoid tending ants so that they are still able to effectively parasitize their host scale insects (e.g. Bartlett 1961, Völkl 1994, 2001; Barzmann and Daane 2001, Kaneko 2007). Some parasitoids have higher rates of parasitism in

5 the presence of ants than in their absence (Völkl and Novak 1997, Tegelaar et al. 2011) and tending ants can even provide "ant-adapted" parasitoids with protection from their natural enemies, including hyperparasitoids (Völkl 1992). Our data indicate that T. somervillei is able to successfully parasitize Tachardina aurantiaca across a variety of host species in the presence of a variety of tending ants, including Anoplolepis gracilipes.

6 TABLES and FIGURES

Table 1.1. Mitochondrial COI base pair sequences of Tachardiaephagus somervillei individuals from a site in East Malaysia (KUCHING, n = 3) and two sites in West Malaysia (FRIM, n = 5 and KLANG, n = 2). A 600 base-pair sequence is shown for each individual. The sequences are identical for this gene fragment across all individuals and sites, indicating that T. somervillei is a single species across this geographic range. Unpublished data 2014, courtesy of Dr Nick Murphy, La Trobe University.

Site Base Pair Sequence

KUCHING_1 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA KUCHING_4 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA KUCHING_9 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA FRIM_16 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA FRIM_19 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA FRIM_20 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA FRIM_21 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA FRIM_3 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA KLANG_2 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA KLANG_5 GATGTATTTATATTACGATCAAATAAAAGTATTGTAATAGCTCCTGCTAATACAGGTAAA

KUCHING_1 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT KUCHING_4 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT KUCHING_9 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT FRIM_16 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT FRIM_19 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT FRIM_20 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT FRIM_21 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT FRIM_3 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT KLANG_2 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT KLANG_5 GATAATAATAATAAAATAGCTGTTAATAATATTGCCCAAGAAAATAAAGGTAAAATCTCT

KUCHING_1 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA KUCHING_4 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA KUCHING_9 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA FRIM_16 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA FRIM_19 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA FRIM_20 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA FRIM_21 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA FRIM_3 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA KLANG_2 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA KLANG_5 ATTTTATATAATTTTATATTAATAATAGTAGTAATAAAATTAATTGAACCTATAATTGAA

KUCHING_1 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT KUCHING_4 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT KUCHING_9 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT FRIM_16 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT FRIM_19 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT

7 FRIM_20 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT FRIM_21 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT FRIM_3 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT KLANG_2 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT KLANG_5 GATAGTCCAGCAATATGAAGAGAAAAAATAGATAAATCTACTGAAGGCCCTATATGAGAT

KUCHING_1 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT KUCHING_4 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT KUCHING_9 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT FRIM_16 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT FRIM_19 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT FRIM_20 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT FRIM_21 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT FRIM_3 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT KLANG_2 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT KLANG_5 AAATTTGAAGATAAAGGAGGGTAGACAGTTCACCCGGTTCCGGTACCTCTACCTACAAAT

KUCHING_1 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC KUCHING_4 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC KUCHING_9 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC FRIM_16 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC FRIM_19 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC FRIM_20 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC FRIM_21 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC FRIM_3 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC KLANG_2 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC KLANG_5 ATCCTAGAAATTAACAAAATAATTCTAGGAGGTAATAATCAAAAACTTATATTATTTATC

KUCHING_1 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC KUCHING_4 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC KUCHING_9 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC FRIM_16 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC FRIM_19 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC FRIM_20 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC FRIM_21 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC FRIM_3 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC KLANG_2 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC KLANG_5 CGAGGAAATACTATATCAGGAGCCCCTATAATTAAAGGAATTAAAAAATTACCAAAACCC

KUCHING_1 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA KUCHING_4 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA KUCHING_9 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA FRIM_16 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA FRIM_19 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA FRIM_20 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA FRIM_21 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA FRIM_3 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA KLANG_2 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA

8 KLANG_5 CCCATCATTACTGGTATTACAAAAAAAAAAATCATTACAAAAGCATGAGCTGTAACAATA

KUCHING_1 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA KUCHING_4 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA KUCHING_9 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA FRIM_16 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA FRIM_19 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA FRIM_20 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA FRIM_21 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA FRIM_3 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA KLANG_2 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA KLANG_5 GAATTATAAATCTGATCATTCCCAATAAAGGAACCAGGAGTCCCTAATTCTAAACGAATA

KUCHING_1 ATTATT KUCHING_4 ATTATT KUCHING_9 ATTATT FRIM_16 ATTATT FRIM_19 ATTATT FRIM_20 ATTATT FRIM_21 ATTATT FRIM_3 ATTATT KLANG_2 ATTATT KLANG_5 ATTATT

9

Table 1.2. Ant attendance of adult females of Tachardina aurantiaca and parasitism rates by Tachardiaephagus somervillei (±SE, N = 5 aggregates of T. aurantiaca at each site) in Peninsular Malaysia, Sarawak and Sabah. Parasitism rates of T. aurantiaca are high in the presence of tending ants, including Anoplolepis gracilipes, on a variety of host plants across the region.

Female Location Site Host plant Tending ants parasitism (%)

Singapore National University Acacia sp. Dolichoderus sp. 73 ± 12

Peninsular Klang (Selangor) Acacia mangium Oecophylla 38 ± 17 Malaysia x A. auriculiformis smaragdina

Tahman Ehsan Milletia pinnata Dolichoderus sp. 46 ± 21 (Selangor) Sarawak Kampung Istana, Acacia mangium Anoplolepis 42 ± 23 Kuching x A. auriculiformis gracilipes

Kampung Boyan, Acacia mangium Oecophylla 81 ± 6 Kuching x A. auriculiformis smaragdina Sabah Sandakan Milletia pinnata Anoplolepis 76 ± 8 gracilipes Sepilok Calliandra Anoplolepis 29 ± 13 haematocephala gracilipes

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Figure 1.1. Tachardiaephagus somervillei, a primary parasite of the yellow lac scale Tachardina aurantiaca in Malaysia and Singapore (female, left; male, right). Males have longer antennae, with whorls of setae. These parasitoids are minute, with a body length of ~2.0 mm and a wingspan of around 3.5 mm (scale bars are 0.5 mm). For scale, one individual fits inside the 0 of the ‘20’ on an Australian 20c piece. Line drawings reproduced from Madhihassan (1957).

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Taiwan

Tachardiaephagus somervillei

Figure. 1.2. Distribution of Tachardiaephagus somervillei (yellow circles) based on records from Mahdihassan 1923, Ferrière 1935, Hayat et al. 2010, G. Neumann, unpublished results). The putative native distribution is indicated by yellow circles and the only likely introduced record by a red circle. A Tachardiaephagus sp., likely to be T. somervillei, established in Taiwan after it was introduced accidentally with lac brood (Kerria lacca) from Thailand in 1940 (Chiu et al. 1985).

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A C

D

B

E

Figure 1.3. A. Adult female of Tachardiaephagus somervillei on Acacia mangium x A. auriculiformis in Kuching, Sarawak. B. Adult female T. somervillei in typical position for oviposition (head and thorax elevated) on the "sidewall" of an adult female of Tachardina aurantiaca. C. Multiple exit holes in individual T. aurantiaca (red arrows), indicating superparasitism by Tachardiaephagus somervillei. D. Parasitism of adult females of Tachardina aurantica by Tachardiaephagus somervillei in the presence of the yellow crazy ant Anoplolepis gracilipes. Arrows indicate discoloured Tachardina aurantiaca, characteristic of parasitized individuals. E. Parasitoid emergence holes in tests of an aggregate of old adult females of T. aurantiaca on Milletia pinnata near Sandakhan, Sabah. Yellow crazy ants (Anoplolepis gracilipes) tended T. aurantiaca at this site. Photos: G. Neumann.

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5

4 T. T. somervillei 3

2 No. ofprogeny ofNo.

1

0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Female T. aurantiaca size (mm)

Figure 1.4. The positive and significant relationship between female size of the yellow lac scale Tachardina aurantiaca and the number of progeny of the parasitoid Tachardiaephagus somervillei emerging from each host scale under field conditions in Kuching, Sarawak, Malaysia. A random sample of 50 parasitized female T. aurantiaca was measured (greatest horizontal diameter of test) and emerging adult parasitoids counted from each host (R2 = 0.471, N = 50 females, p < 0.01).

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SECTION 2 An assessment of both the possible direct impacts and consequential impacts of the action to the environment, including people and communities, flora and fauna species, the rainforest ecosystem and Ramsar wetlands. This should include consideration of the likely ecological niche of T. somervillei in the Christmas Island ecosystem over time, including predation by and on T. somervillei, competition from T. somervillei with other species, the introduction of pathogens or hyperparasitoids and parasitization of non-target species. If appropriate, provide details of any proposed management measures to be implemented to mitigate any potential impacts.

[Some of these issues were addressed in the original Environmental Referral (EF Part 2.1 Approval, Importation & Rearing, and Release & Monitoring of Tachardiaephagus, pp. 12-13; EF Part 4 Measures to avoid or reduce impacts, pp. 33-34; RA Part A Identifying and Analysing Project Management Risks a. Risk Ref/ID1, p. 21-22, b. Risk Ref/ID2, p. 22, c. Risk Ref/ID3, p. 23, d. Risk Ref/ID4, p. 23-24, e. Risk Ref/ID5, p. 24; and, RA Part B Risk Register & Mitigation - Risk ref/IDs 1-5, pp. 25-27; and in the addendum to the Environmental Referral supplied to the EACD (Amendments_Referral_2013_6836_2May2013.docx)]. The most likely direct impact of the importation, release, establishment and population build-up of Tachardiaephagus somervillei is the population decline of its known host, the introduced and invasive yellow lac scale Tachardina aurantiaca, on a variety of host plants in rainforest and in settled areas (T. aurantiaca is a broad host plant generalist and attacks at least 35 native species in 22 plant families in island rainforest as well as at least 11 introduced species in eight families of fruit trees and amenity plants in Settlement). In both settings, suppression of T. aurantiaca by Tachardiaephagus somervillei would reduce impacts (i.e., improve survival, growth and production) on affected plant species. Even more importantly, suppression of T. aurantiaca by T. somervillei should reduce honeydew availability to the invasive yellow crazy ant Anoplolepis gracilipes so as to lead to the decline of high-density supercolonies (Green et al. 2013) and reduction in the wide variety of known, unwanted impacts they have on the environment on Christmas Island (e.g. O'Dowd et al. 2003; Davis et al. 2008, 2010; Green et al. 2011), including Ramsar wetlands, listed threatened species and communities, listed migratory species, flora and fauna species, and people and communities.

Comments on relevant controlling provisions for the proposed action: A. Wetlands of international importance (Sections 16 & 17B of the Act). The principal threat to the two Ramsar wetland of international significance on Christmas Island, Hosnies Spring (202 ha) and The Dales (580 ha), is the impact of high-

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density supercolonies of the yellow crazy ant (The Wetlands Policy of the Commonwealth Government of 1997). The risk to the Ramsar wetlands posed by the yellow crazy ant not only involves the direct effects of predation and competition but also the negative impacts of its mutualists, especially the yellow lac scale (Tachardina aurantiaca). Canopy dieback is apparent in supercolonies of the yellow crazy ant where high densities of scale insects occur (O'Dowd et al. 2003). The levels of these effects vary among tree species. Seedlings, saplings, and trees of one of the canopy dominants in these Ramsar wetlands, the Tahitian Chestnut (Inocarpus fagifer), suffer extremely high mortality as a result of infestation by the yellow lac scale. Furthermore, excess honeydew that yellow crazy ants do not harvest settles on leaves of all plant species and is colonized by sooty moulds, which probably interferes with photosynthesis and growth (O’Dowd et al. 2003, P. Green and D. O’Dowd, unpublished results). The proposed actions in this referral are directed at mitigating the impacts of high- density supercolonies of the yellow crazy ant on all three key criteria (criteria 1,3 and 4) on which listing of Hosnies Spring was based and three of five criteria (criteria 1, 3 and 4) that led to the listing of The Dales (see http://www.environment.gov.au/node/33425 for descriptions of these criteria). Supercolonies of the yellow crazy ant have formed across most of the Hosnies Spring and The Dales wetlands over the past 12 years, respectively (Fig. 2.1). Current management activities, involving hand-baiting or aerial distribution of Presto 01, a bait with the active ingredient as Fipronil (a general invertebrate intoxicant) have been taken across the supercolonies (i.e. three aerial baiting campaigns and more limited hand- baiting, encompassing a total of over 55 and 77 percent of the total area of Hosnies Spring and The Dales, respectively). Furthermore, fipronil, because of its toxicity in aquatic ecosystems, cannot be used in the “core” area of the wetlands to suppress these supercolonies (52 ha at Hosnies Spring and 137 ha at The Dales, respectively). Sustained suppression of high-density supercolonies of the yellow crazy ant by reduction of the honeydew supply following biological control of Tachardina aurantiaca by the parasitoid Tachardiaephagus somervillei would maintain key criteria that led to the listing of both sites as Ramsar wetlands of international importance.

B. Listed threatened species and communities (Sections 18 & 18A of the Act).

The yellow crazy ant Anoplolepis gracilipes is listed as a Key Threatening Process on Christmas Island under the EPBC Act 1999 (http://www.environment.gov.au/node/14577). Furthermore, the Expert Working Group (Beeton et al. 2010) concluded that the yellow crazy and scale insect association is the key factor generating the threat. Twelve of the 16 EPBC Act listed species are given as threatened by yellow crazy ants on Christmas Island (Tables 2.1 and 2.2) The most likely direct impact of the importation, release, establishment and population

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build-up of Tachardiaephagus somervillei is the population decline of the introduced and invasive yellow lac scale Tachardina aurantiaca. This in turn should reduce formation of high-density supercolonies of the introduced and invasive yellow crazy ant so as to mitigate its known or inferred impact on listed species and other species of conservation concern. Of the EPBC Act listed species given as threatened by yellow crazy ants on Christmas Island, there is strong empirical evidence that two species, the Christmas Island thrush (Turdus poliocephalus eythropleurus) and the Christmas Island Emerald dove (Chalcopaps indica natalis) are negatively affected in yellow crazy ant supercolonies (Davis et al. 2008). This evidence was central to the decision to list them as endangered. More broadly, supercolony formation by the yellow crazy ant rapidly changes habitat structure, plant recruitment dynamics, litter dynamics, and resource availability in rainforest on Christmas Island (O'Dowd et al. 1999, 2003; Davis et al. 2008; Green et al. 2011). These changes may affect other listed species. Supercolonies of the yellow crazy ant have been implicated in the declines of critically endangered species (e.g., Pipistrellus murrayi, Cryptoblepharus egeriae), and other endangered species (e.g., Cyrtodactylus sadlieri) (Beeton et al. 2010). Although no communities on Christmas Island are currently listed as threatened under the EPBC Act, it is well documented that the sustained formation of yellow crazy ant supercolonies over hundreds of hectares of rainforest results in rapid and persistent changes in forest structure and diversity as well as ecosystem processes (O'Dowd et al. 1999, 2003; Green et al. 2011). The superabundance of Tachardina aurantiaca on preferred plant host species where high-density supercolonies of the yellow crazy ant have formed (Abbott and Green 2007) has decreased plant growth and reproduction, and led to canopy dieback, mass mortality and recruitment failure of some dominant canopy tree species, including Inocarpus fagifer, in island rainforest (O'Dowd et al. 2003, P. Green and D. O'Dowd, unpublished results). The most likely direct impact of the importation, release, and build-up of populations of Tachardiaephagus somervillei is the reduction in the density of their host Tachardina aurantiaca and the mitigation of its impacts on especially susceptible host plant species including I. fagifer, Milletia pinnata, and Tristiropsis acutangula. Since none of the over 22,000 described species of chalcidoid parasitoids, including the approximately 3,700 species of encyrtid parasitoids, is known to attack vertebrates anywhere in the world (see Noyes 2014), the likelihood of any direct negative effect of T. somervillei on any listed threatened species is negligible.

C. Listed migratory species (Sections 20 & 20A of the Act)

By suppressing populations of Tachardina aurantiaca and the formation of high-density supercolonies of the yellow crazy ant, the most likely and consequential impacts of the importation and release of Tachardiaephagus somervillei on Christmas Island is the

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reduction in any impact of the yellow crazy ant on listed migratory species (see Appendix E, Director of National Parks 2014a). Since none of the over 22,000 described species of chalcidoid parasitoids, including the approximately 3,700 species of encyrtid parasitoids, is known to attack vertebrates anywhere in the world (see Noyes 2014), the likelihood of any direct negative effect of T. somervillei on any listed migratory species is negligible.

D. Flora and fauna species

For flora species, the most likely direct impact of the importation, rearing, release, and population build-up of Tachardiaephagus somervillei is the suppression of the invasive yellow lac scale and a decrease in the impacts it has on at least 35 native rainforest species in 22 plant families on Christmas Island (Table 2.3). This is especially so for Inocarpus fagifer, Tristiropsis acutangula, Milletia pinnata, and Terminalia catappa, important tree species that suffer dieback and high mortality from infestation by Tachardina aurantiaca. More broadly, the primary aim of the importation, rearing, release, establishment and population build-up of T. somervillei is the indirect suppression of yellow crazy ant supercolonies and restoration of population densities of the red land crab in rainforest areas affected directly and indirectly by high-density supercolonies of the yellow crazy ant. Through the reduction of honeydew production by Tachardina aurantiaca, high- density supercolonies should be suppressed and their impacts on flora and fauna reduced. Although the red crab, a keystone species, and other endemic land crabs remain unlisted, they are considered as Significant Species (Table 2.2, sensu Draft Christmas Island Conservation Plan, Director of National Parks 2014). The clearest and best-documented impact of high-density supercolonies of the yellow crazy ant is on these species, especially the red land crab Gecarcoidea natalis. Furthermore, the extirpation of the red land crab Gecarcoidea natalis in high-density supercolonies and population decline in areas outside of supercolonies of the yellow crazy ant affected through 'ghosting' (O'Dowd et al. 2003, Green et al. 2011) are the primary forces generating broader changes in habitat structure and resource levels that affect listed and other important flora and fauna species and communities on Christmas Island (O’Dowd et al. 1999, 2003; Beeton et al. 2010, Green et al. 2011, Director of National Parks 2014b).

E. People and communities

The most likely direct impact of the importation, rearing, release, and population build- up on peoples and communities on Christmas Island is the restoration and sustainability of biodiversity, amenity and cultural heritage values as related to rainforest on Christmas Island. As one brief example to illustrate the amenity and cultural value of rainforest, the Ramsar wetland at The Dales (see Section 2A above), is an area of both

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local recreation and cultural significance. The CI Rey Tseng Temple Association has declared the Hugh's Dale waterfall as a significant cultural site in relation to their worship, and regularly conducts ceremonies and worship at the site (Director of National Parks 2014a). More broadly, by reducing broad impacts on the assemblages of Significant Species (sensu Draft Christmas Island Biodiversity Conservation Plan, Director of National Parks 2014), the proposed action detailed in the Environmental Referral could facilitate sustainable nature-based tourism and recreational benefits so as to broaden the economic base of the island. There is no possibility that T. somervillei, a minute chalcidoid wasp ~2 mm in length, could directly harm humans or interfere with human activities in island rainforest. T. somervillei is a solitary "microwasp," not a social wasp or a generalist predator, and it does not sting or show any aggressive behaviours to humans. Furthermore, unlike large colonial, social wasps, they cannot and do not form nests in gardens or houses. In gardens in Settlement on Christmas Island, a variety of microwasp species (see Section 4, Table 4.2), introduced during human settlement on the island, that attack other introduced scale insects are already present and considered as beneficial insects. In Settlement on Christmas Island, the most likely impact of importation and release of T. somervillei is a direct negative impact on the yellow lac scale Tachardina aurantiaca, which could improve growth and production of some valued horticultural and amenity plants (Table 2.2).

F. Likelihood of niche expansion of the proposed biological control agent, Tachardiaephagus somervillei.

The risk of the host range of T. somervillei expanding beyond species in the Kerriidae, the lac scale insects, to other families in the Superfamily Coccoidea is remote, based on both historical records (Noyes 2014) and our detailed host specificity testing in the area of origin with positive controls (see Section 3 below for details). Furthermore, no known encyrtid parasitoid that attacks lac scale insects (Kerriidae) has a host range that extends beyond superfamily Coccoidea. Importantly, there are no endemic scale insect species (Coccoidea) on Christmas Island; all are introduced exotics (see Section 3). One other kerriid species, Paratrachardina pseudolobata Kondo & Gullan, has been introduced to Christmas Island and there is one record of Tachardiaephagus tachardiae, closely related to T. somervillei, emerging from a congeneric species, P. silvestri in India (Varshney et al. 1967). Thus, it is possible that T. somervillei could also use P. pseudolobata as a host species on Christmas Island. However, since P. pseudolobata attacks a wide variety of native rainforest plant species and introduced fruit trees that occur on Christmas Island (Abbott 2004; Howard et al. 2006; Pemberton et al., unpublished results), it is difficult to envisage that use of this introduced and invasive lac scale species by T. somervillei as a host as anything but a direct positive impact resulting from the importation, release, and population build-up of T. somervillei.

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The prospect that T. somervillei's host range could expand even more broadly to include any known endemic insect, listed threatened species, or listed migratory species on Christmas Island is even more remote (see Section 3 below). All listed species are in a different Phylum (Vertebrata) than the hosts of T. somervillei (Arthropoda: Insecta: Hemiptera: Kerriidae) and there is not a single case in the global literature of any one of the approximately 22,000 described species of chalcidoid parasitoids attacking any vertebrate species (Noyes 2014).

G. Predation by or of Tachardiaephagus and competition with other species.

No listed threatened or migratory species, or other fauna species on Christmas Island appears dependent on the superabundant, invasive lac scale Tachardina aurantiaca as an important prey item. In Labuan, Sabah in Malaysia, the Sunda pygmy woodpecker (Dendrocopos moluccensis) has been observed pecking aggregates of Tachardina aurantiaca (C.L. Liung, personal communication 2013), and on Christmas Island, the endemic Christmas Island white-eye Zosterops natalis, an insectivore/frugivore, has also been observed pecking adult females of T. aurantiaca in yellow crazy ant supercolonies (Davis et al. 2008). However, these few feeding observations and the rarity of bird- damaged tests of T. aurantiaca indicate that it is not an important food item. The Christmas Island white-eye could conceivably collect honeydew produced by T. aurantiaca, but this was not observed during a five-month study of Z. natalis and other endemic bird species in supercolonies of the yellow crazy ant (N. Davis, pers. comm. 2011). Parasitization by T. somervillei is restricted to hosts in the Kerriidae (see Section 3 below). All other remotely feasible hosts are introduced scale insects, so there is no likelihood of direct competition with any parasitoid species that attacks any native or endemic insect species on Christmas Island. Marietta leopardina (), the only parasitoid of Tachardina aurantiaca already present on Christmas Island, should complement the effects of T. somervillei, since it attacks only male Tachardina while T. somervillei attacks only mature females. In its native range, the most important natural enemies of T. somervillei are other microhymenopteran parasitoid species that are either obligatory or facultative hyperparasitoids (Fig. 2.2A). There, these natural enemies could be abundant enough in some instances to regulate population densities of T. somervillei. No true predators of Tachardiaephagus somervillei or other Tachardiaephagus spp. are known, although larval stages parasitizing Tachardina aurantiaca could be consumed incidentally by both predatory insects (e.g. lac-feeding specialists like the predaceous lepidopteran Eublemma spp. and generalist predators like neuropteran Chrysopa spp.) or insectivorous birds, and it is possible that adult Tachardiaephagus somervillei searching for suitable hosts or feeding on honeydew might be captured by the introduced ants that tend T. aurantiaca on Christmas Island. However, the high levels of parasitism of Tachardiaephagus somervillei seen in Malaysia (see Table 1.2) indicate that any

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predation by ants tending Tachardina aurantiaca is unlikely to regulate the population of this parasitoid.

H. Potential impacts of co-introduction of hyperparasitoids of Tachardiaephagus somervillei and phyosanitary procedures to minimize risk.

In its native distribution in Malaysia, Tachardina aurantiaca supports a complex suite of natural enemies, including specialized predators, primary parasitoids (e.g. Tachardiaephagus somervillei), and hyperparasitoids of primary parasitoids (Fig. 2.2A). Three facultative hyperparasitoids are known to attack T. somervillei or related T. tachardiae. Two species, Promuscidea unfasciativentris (Aphelinidae) and purpureus (Eulophidae)(Fig. 2.2A) are both primary parasites of a variety of scale insects (Coccoidea) and hyperparasites of a variety of encyrtid, pteromalid, and aphelinid parasitoids (Noyes 2013). Marietta leopardina (Aphelinidae), also a facultative hyperparasitoid, is a primary parasitoid known from Coccoidea in eight families, two species from the (Suborder ), one species of Cicadellidae () two species of Cecidomyiidae (Diptera), and one species of (Noyes 2012). In its area of introduction on Christmas Island, few of these or other natural enemies occur (Fig. 2.2B) and none in sufficient abundance to potentially regulate the population of Tachardina aurantiaca (Green et al. 2013). Of the parasitoids, only Marietta leopardina is present (Fig. 2.2B), almost certainly introduced inadvertently from Southeast Asia in association with scale insect hosts on transported plant material during human settlement of the island. On Christmas Island, it is known only as a primary parasitoid of male T. aurantiaca (Green et al. 2013). Inadvertent importation of a hyperparasite of T. somervillei could be inimical to its successful suppression yellow crazy ant supercolonies because it could compromise the capacity of Tachardiaephagus to build up population densities sufficient to control Tachardina. For this reason, great care will be taken to implement standard agent rearing and sanitary techniques to ensure that the founding population of Tachardiaephagus somervillei from Malaysia is free of hyperparasitoids. Free-living adults of T. somervillei are the safest to import because this would ensure that hyperparasitoids would not be accidentally co-introduced.

I. Protocol for insuring that hyperparasitoids associated with T. somervillei are not imported to Christmas Island

Tachardiaephagus somervillei is frequently attacked by Promuscidea unfasciativentris (Aphelinidae) in its native distribution (Fig. 2.2A). This hyperparasitoid was abundant at sites near Kuching, Sarawak and present in Selangor, West Malaysia, although rare. Although the impact of hyperparasitism on populations of T. somervillei in its native distribution is not known (parasitism rates of field populations of Tachardina aurantiaca

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by Tachardiaephagus somervillei can still be very high in the presence of hyperparasitoids – see Table 1.2), the exclusion of any hyperparasitoids from captive colonies in Malaysia is critical before adult T. somervillei are imported to Christmas Island as the founding population.

The exclusion of hyperparasitoids from captive populations at locations where the hyperparasitoids are native can be difficult unless care is taken. Standard practices to keep hyperparasitoids out of captive populations will be followed. These steps include:

1. Field-collected, parasitized Tachardina will never come into contact with laboratory Tachardina used for rearing Tachardiaephagus somervillei. Field-collected stems with parasitized scale insects will be placed into emergence cages in a separate facility from the rearing facility. 2. Emerging parasitoids will be examined individually under magnification before being removed from emergence cages. The range of hyperparasitoids of T. somervillei is known (Fig. 2.2A) and all have distinctive morphologies from T. somervillei. If hyperparasitoids (or any organism other than T. somervillei) are found in any emergence cage, the cage will be removed and destroyed. 3. Emergence cages will be kept in a dedicated room (the emergence room, see below) in a separate building from the rearing facility (a dedicated room where the captive lac scale Tachardina and parasitoid T. somervillei population is located. 4. T. somervillei will be moved to the rearing facility and placed on host plants with suitable scale insect hosts in a fine mesh bag; the mesh bag ensures that parasitoids remain on the plant and the scale insects are protected. 5. Mesh bags will not be removed (only shortly for monitoring purposes) from the plants until parasitoid emergence. 6. When parasitoid emergence is expected, host plants with parasitized scales in their mesh bags will be moved to the emergence room. 7. Personnel conducting field collections or any other field activity will not be allowed in the rearing facility on the same day.

It is critical that the captive population is monitored continuously for the presence of hyperparasitoids even if best practices are followed closely. In addition to individual examinations of parasitoids, yellow sticky cards will be placed in both the emergence room and the rearing facility to monitor (and also trap) any hyperparasitoids or other insects. Controlled exposures inside mesh bags will protect T. somervillei.

The elimination of hyperparasitoids before importation of adult T. somervillei to Christmas Island will involve the following steps.

1. The sanitary practices followed during emergence and rearing (see above) will ensure a "clean" captive population. 2. Emerged, adults of Tachardiaephagus somervillei will be placed in airtight, glass

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vials. All individual parasitoids will be inspected under magnification prior to packing. No Tachardina hosts will leave the rearing facility. 3. Glass vials with adult parasitoids will be packed in a sealed travel cooler to buffer them from extremes of temperature in transit. This will be hand-carried from the rearing facility at the Forest Research Institute Malaysia in to Christmas Island . 4. All individuals will be examined again under magnification at the point of entry on Christmas Island while still in the glass vials. Training will be provided in advance for quarantine personnel on Christmas Island to identify T. somervillei and differentiate it from any other parasitoid(s) or other organism(s). If in any doubt, the vial containing the questionable organism(s) will be destroyed. 5. All individuals will be inspected again in the rearing facility on Christmas Island before the parasitoids are removed from the glass vials.

In practice, the above procedures will ensure that hyperparasitoids of T. somervillei are not transported to Christmas Island from Malaysia.

Inadvertent introduction of entomopathogens from Malaysia that affects T. somervillei might also be inimical to its successful establishment and population buildup once introduced to Christmas Island. There are no records of any pathogens affecting T. somervillei and our observations in Kuching (Sarawak) and Kuala Lumpur suggest that pathogens do not play a significant role in natural and laboratory populations of T. somervillei. Based on external inspection and dissections, not a single T. somervillei, either field collected or laboratory reared in Malaysia, showed any signs of infection by entomopathogenic fungi or nematodes.

Entomopathogenic fungi and nematodes are probably not significant risk factors, but microsporidia (protozoan pathogens) can cause losses in fitness and affect searching ability of parasitoids after establishment from field populations (e.g. Geden et al. 2003). Since these pathogens can be transmitted vertically to the progeny, it is important to monitor all subsequent generations for the presence of spores. Although there is no evidence of microsporidia in T. somervillei, the parental generation and their progeny will be monitored for such infection, if any, in samples of parental generation and progeny. The presence of spores can be easily detected by macerating the full body of individual parasitoids, mixing the sample with distilled water, transferring the sample onto a microscope slide and examine using simple light microscopy (stained or unstained samples) at 100x or 400x magnification.

J. Importation of a founder population of Tachardiaephagus somervillei from Malaysia to Christmas Island.

The transfer of the founder population from Malaysia to Christmas Island could be as simple as packaging freshly emerged adult T. somervillei inside glass vials that are placed

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inside a sealed travel cooler. The adult parasitoids survive well without access to food or water for 48 hours but provision of sugar solution and water increases survival to several weeks (see Section 1E above). If parasitoids are likely to be in transit for longer than two days, a sugar solution and water will be provided within the glass vials.

Currently, there are two possible pathways to transport the founder population to Christmas Island. First, direct flights are scheduled weekly between International Airport and Christmas Island, and the founder population could be transported from Kuala Lumpur International Airport to Christmas Island via Jakarta. This assumes that while the founder population is in transit at Jakarta, Indonesian quarantine regulations can be satisfied. Alternatively, the founder population could be transported directly from Kuala Lumpur to International Airport, placed in quarantine overnight, released from quarantine and then transported directly to Christmas Island the next day. This would need to comply with Department of Agriculture regulations.

An experienced biocontrol professional, Dr. Gabor Neumann (La Trobe University, [email protected]) will supervise the production of the founder parasitoid population in Malaysia, ensure that the insects are free of entomopathogens and hyperparasitoids, hand carry the founder population to Christmas Island, and supervise rearing in the rearing facility on Christmas Island. Dr. Neumann has previously conducted foreign exploration for natural enemies, determination of biology and life cycle of the potential biological control agent, and host specificity testing of the potential biological control agent.

K. Risks

The risk imposed upon species identified as Significant on Christmas Island by the introduction of the host-specific parasitoid Tachardiaphagus somervillei on Christmas Island is negligible (see analyses in Section 3A and 3B). None of the approximately 3700 species of Encyrtidae described over the past 200 years (Noyes 2014), including T. somervillei, has ever been reported to use a vertebrate (Phylum Vertebrata) or plant (Kingdom Plantae) as a host. Consequently, the risk of "jumping hosts" to any of the species listed in Table 2.2 is remote.

The introduction of a biological control agent from its area of origin, just like any other deliberate introduction of a species for whatever reason, is usually difficult or impossible to recall. This is the reason that well-defined protocols and strict regulatory frameworks have been developed and used to evaluate risk prior to any decision to introduce an agent in many countries and regions (e.g. the United States, Canada, the European Union, Australia, New Zealand, and Japan). In Australia, the legislative regulatory framework is conservative and uses very much a precautionary approach. The production of an Import Risk Analysis for a biological control agent is based entirely

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on an evaluation of the risks posed; potential benefits are not considered (http://www.agriculture.gov.au/ba/ira/process-handbook.)

The likely consequences of failing to manage supercolonies of the yellow crazy ant and associated scale insects are very large. They are given to affect at least 12 of 16 EPBC Act listed species and other species of concern that are internationally iconic or play critical roles in determining rainforest structure and dynamics on the island.

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TABLES and FIGURES

Table 2.1. EPBC Act listed species that list yellow crazy ants as a threat (P. 29, 'Novel biota and their impacts on biodiversity' listing advice to the Minister from the Threatened Species Scientific Committee, http://www.environment.gov.au/node/14591).

Scientific name Common name EPBC Act listing

Accipiter hiogaster natalis Christmas Island Endangered goshawk

Chalcophaps indica natalis Emerald dove Endangered (Christmas Island)

Chelonia mydas Green turtle Vulnerable

Crocidura attenuata trichura Christmas Island shrew Endangered

Eretmochelys imbricata Hawksbill turtle Vulnerable

Fregata andrewsi Christmas Island Vulnerable frigatebird

Lepidodactylus listeri Christmas Island gecko Vulnerable

Ninox natalis Christmas Island hawk- Vulnerable owl

Papasula abbotti Abbott's booby Endangered

Pipistrellus murrayi Christmas Island Critically Endangered pipistrelle

Ramphotyphlops exocoeti Christmas Island blind Vulnerable snake

Turdus poliocephalus Island thrush Endangered erythropleurus (Christmas Island)

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Table 2.2. Species of Christmas Island currently identified as Significant* (Director of National Parks 2014b).

Significance Species criteria ccriteriariteria Vascular plants Asplenium listeri Christmas Island spleenwort 1 Bruguiera gymnorhiza, mangroves 2 B. sexangula Pneumatopteris truncata a fern 1 Tectaria devexa var. minor a fern 1

Seabirds Fregata andrewsi Christmas Island frigatebird 1,4 Papasula abbotti Abbott’s booby 1,4 Phaethon lepturus fulvus golden bosun, white-tailed tropicbird 4

Forest birds Accipiter hiogaster natalis Christmas Island goshawk 1,4 Chalcophaps indica natalis Christmas Island emerald dove 1,4 Collocalia linchi natalis Christmas Island swiftlet 4 Ducula whartoni Christmas Island imperial pigeon 2,4 Ninox natalis Christmas Island hawk-owl 1,4 Turdus poliocephalus Christmas Island thrush 1,4 erythropleurus Zosterops natalis Christmas Island white-eye 2,4

Mammals Crocidura trichura Christmas Island shrew 1,4 Pipistrellus murrayi Christmas Island pipistrelle 1,4 Pteropus melanotus natalis Christmas Island flying-fox 1,2,4

Reptiles Cryptoblepharus egeriae blue-tailed skink 1,4 Cyrtodactylus sadleiri giant gecko 1,4 Emoia atrocostata coastal skink 3 Emoia nativitatis forest skink 1,4 Lepidodactylus listeri Lister’s gecko 1,4 Ramphotyphlops exocoeti Christmas Island blind snake 1,4

Land crabs Birgus latro robber crab 5 Discoplax celeste blue crab 2 Gecarcoidea natalis red crab 2,3,5

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*For significance criteria, 1 = a species listed or threatened under the EPBC Act, 2 = a species with an important or 'keystone' role in maintaining the island's ecology or which characterizes a significant ecosystem; 3 = species which are of conservation concern but not listed as threatened, 4 = an endemic vertebrate, and 5 = a species of international conservation significance.

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Table 2.3. Host plant species of the yellow lac scale Tachardina aurantiaca (Hemiptera: Kerriidae) on Christmas Island, arranged alphabetically by family. Nomenclature follows that used in (http://www.theplantlist.org/). For host location, Christmas Island National Park (CINP) or the Northeast settled part of the island (Settlement).

Family Species Host location Growth form Source

Boraginaceae Ehretia microphylla CINP shrub Abbott 2004 Lam. Cannabaceae Celtis timorensis Span. CINP tree Abbott 2004

Combretaceae Terminalia catappa L. CINP tree Abbott 2004

Euphorbiaceae Acalypha indica L. Settlement shrub Pemberton & O'Dowd, pers. obs.2007 Claoxylon indicum CINP tree Abbott 2004 (Reinw. ex Blume) Hassk. Croton caudatus Geisler CINP vine Abbott 2004 Macaranga tanarius (L.) CINP tree Abbott 2004 Müll.Arg. Hernandiaceae Hernandia ovigera Lam. CINP tree Abbott 2004 Lamiaceae Callicarpa longifolia CINP shrub Abbott 2004 Lam. Leucas decemdentata CINP herb Abbott 2004 (Willd.) Sm. Premna lucidula Miq. CINP tree Green & O'Dowd, pers. obs. Lauraceae Cryptocarya nitens CINP tree Abbott 2004 Koord. & Valeton Lecythidaceae Barringtonia racemosa CINP tree Abbott 2004 (L.) Spreng. Leguminosae Cynometra ramiflora L. CINP tree Abbott 2004 Erythrina variegata L. CINP tree Abbott 2004 Inocarpus fagifer CINP tree Abbott 2004 (Parkinson) Fosberg Milletia pinnata (L.) CINP tree Abbott 2004; G. Panigrahi Neumann, pers. obs. 2013 Lythraceae Punica granatum L. Settlement tree Pemberton & O'Dowd, pers. obs. 2007 Malvaceae Berrya cordifolia CINP tree Green & O'Dowd, (Willd.) Burret pers. obs. Kleinhovia hospita L. CINP tree Abbott 2004 Meliaceae Dysoxylum CINP tree Abbott 2004 gaudichaudianum (A. Juss.) Miq. Moraceae Ficus microcarpa L.f. CINP tree Abbott 2004

Ficus saxophila Blume CINP tree Abbott 2004 Maclura CINP vine Abbott 2004 cochinchinensis (Lour.) Corner Myrtaceae Psidium guajava L. Settlement tree Pemberton & O'Dowd, pers. obs.

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2007 Syzygium nervosum CINP tree Abbott 2004 A.Cunn ex DC.

Syzygium Settlement tree Pemberton & samarangense (Blume) O'Dowd, pers. obs. Merr. & L.M.Perry 2007 Orchidaceae Corymborkis veratrifolia CINP herb O'Dowd, pers. obs. (Reinw.) Blume Oxalidaceae Averrhoa bilimbi L. Settlement tree Pemberton & O'Dowd, pers. obs. 2007 Averrhoa carambola L. Settlement tree Pemberton & O'Dowd, pers. obs. 2007 Primulaceae Ardisia colorata Link CINP tree Abbott 2004 Proteaceae Macadamia integrifolia Settlement tree Pemberton & Maiden & Betche O'Dowd, pers. obs. 2007 Rubiaceae Aidia racemosa (Cav.) CINP shrub Abbott 2004 Tirveng. Rutaceae Acronychia trifoliata CINP tree Abbott 2004 var. trifoliata Zoll. & Moritzi Citrus aurantifolia Settlement shrub Pemberton & (Christm.) Swingle O'Dowd, pers. obs. 2007 Citrus hystrix DC. Settlement shrub Pemberton & O'Dowd, pers. obs. 2007 Sapindaceae Allophylus cobbe (L.) CINP tree Abbott 2004 Raeusch. Dimocarpus longan Settlement tree Pemberton & Lour. O'Dowd, pers. obs. 2007 Dimocarpus longan var. Settlement tree Pemberton & malesianus Leenh. O'Dowd, pers. obs. 2007 Tristiropsis acutangula CINP tree Abbott 2004 Radlk.

Planchonella duclitan CINP tree Abbott 2004 (Blanco) Bakh.f. Solanaceae Capsicum annuum L. Settlement herb Pemberton & O'Dowd, pers. obs. 2007 Solanum melongena L. Settlement herb Pemberton & O'Dowd, pers. obs. 2007 Thymeleaceae Dendrocnide peltata CINP tree Abbott 2004 (Blume) Miq. Urticaceae Leea angulata Korth. Ex CINP tree Abbott 2004 Miq.

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Figure 2.1. Occurrence of yellow crazy ant supercolonies and baiting in the two Ramsar wetlands, Hosnies Spring (top) and The Dales (bottom), on Christmas Island. Total area of supercolony formation across the two wetlands is equivalent to the area baited between 2000-2013.

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Figure 2.2. Food web interactions centred on Tachardina aurantiaca (♀, left; ♂right) in (A) its area of origin (Malaysia, Sundaland) and (B) in its area of introduction on Christmas Island, . In Malaysia, food web interactions are complex involving two predaceous noctuid moths, Eublemma sp. (1) and Holcocera sp. (2), three primary parasitoids, including the potential biological control agent, Tachardiaephagus somervillei (3, stippled circle), Coccophagus tschirchii (4), Marietta leopardina (5), which attacks only males of T. aurantiaca, and two facultative hyperparasitoids, Aprostocetus purpureus (6) and Promuscidea unifasciativentris (7) that attack either Tachardiaephagus somervillei and Coccophagus tschirchii as well as Tachardina aurantiaca. In contrast, on Christmas Island, only three natural enemies of T. aurantiaca are known (1, 2, and 5), and all at low densities that do not affect population densities of T. aurantiaca.

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SECTION 3 Inference of host specificity of the potential biological control agent Tachardiaephagus somervillei

A. Database analysis of host specificity and potential for non-target impacts The genus Tachardiaephagus comprises seven described species distributed in Southeast and South Asia (T. tachardiae, T. somervillei, and T. sarawakensis - Ferrière 1928, 1935, Hayat et al. 2010) and sub-Saharan Africa (T. absonus, T. gracilus, T. similis, and T. communis - Prinsloo 1977). Known host species records for all species were evaluated using the Universal Chalcidoidea Database (Noyes 2012) so that host range could be estimated and risk of release of T. somervillei assessed for listed species and communities, flora and fauna species, listed migratory species, populations and communities on Christmas Island (see Sands and Van Driesche 2004). The Universal Chalcidoidea Database (Noyes 2012) is the most comprehensive database for chalcidoid parasitoids, with over 120,000 host/associate records (including associations with food plants of the hosts) and >140,000 distribution records of the parasitoids in the superfamily Chalcidoidea. It is very well developed, regularly updated and extremely well referenced. Nevertheless, large databases can contain errors affecting reliability, such as erroneous published host records and outdated parasitoid taxonomy (Kuhlmann et al. 2006). The most important source of error in this database is actual published erroneous records. Since all records are referenced, any doubtful records can be investigated and if deemed appropriate, filtered out. The most obvious non-target group on Christmas Island is other scale insects (Superfamily Coccoidea: Sternorryhncha). We used the database to assess the likely host breadth of Tachardiaephagus somervillei, and its congeners and confamilials to evaluate non-target risks to other scales species on the Island. Much less likely is that the introduction of Tachardiaephagus somervillei could pose a risk to much more phylogenetically distant, endemic (non-scale insect) hemipterans (e.g. Suborders Auchenorryhnca and ). We used the database to consider the host breadth of encyrtid parasitoids known to attack species in hemipteran families with known endemics on Christmas Island, as a way of inferring whether the host range of any known encyrtid parasitoid encompasses both scale insects and the families to which the endemic hemipterans belong. Host breadth of T. somervillei, its congeners, and confamilials. Host records show that T. somervillei is only recorded from two host genera (Kerria and Tachardina) in the Kerriidae (Table 3.1) while host records for the remaining six species in the genus add only one more host genus, Paratachardina lobata (= P. silvestri), from which Varshney et al. (1967) reared one T. tachardiae)(Table 3.1). There is a single record of the African Tachardiaephagus similis from the coccid scale insect eucleae, but this was discounted as erroneous by Prinsloo (1977). These records suggest that even at the generic level, Tachardiaephagus is a narrow host specialist on scale insects in the family Kerriidae. A broader analysis of host records for 40 encyrtid species in 16 genera known to attack Kerriidae indicated that most (32/40, 80%) are restricted to hosts within the Kerriidae, and that the host range of the remaining 8 species is restricted to the Superfamily Coccoidea (Table 3.2). This strongly suggests that introduction of any encyrtid parasitoid that attacks host taxa within the Kerriidae would be unlikely to attack any hosts other than scale insects.

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Records for occurrence of scale insect species (Superfamily Coccoidea, the same superfamily to which Tachardina aurantiaca belongs) on the island were compiled from the literature and Scalenet (Ben-Dov et al. 2014). These were supplemented by conducting >400 hours of structured search in all major forest types over three years for endemic scale insects (Green et al. 2013) along with targeted search over three years on 14 of the 18 endemic plant species known on Christmas Island (Table 3.3). A total of 27 species of scale insects in seven families, incorporating both ‘neococcid’ and ‘archeococcid’ taxa (sensu Gullan and Cook 2007) have been recorded from Christmas Island (see Table 4.1, Section 4). Although Tachardiaephagus species appear to be kerriid specialists, a few encyrtid species in other genera have host ranges that encompass other scale insects (see above). Although unlikely, the introduction of T. somervillei could pose a risk to other scale insect taxa (e.g. ), but no endemic scale insects were found in over 400 hours of structured search as well as targeted search of endemic plants over three years. None of the scale insect species on Christmas Island are endemic or likely to be native. Three lines of evidence indicate that the entire scale insect assemblage on Christmas Island is synthetic and has been introduced since human settlement over 125 years ago as a by-product of the unregulated movement of plant materials to the island. First, the areas of origin for these scale insects species (see Miller et al. 2005) are diverse and include all major biogeographic regions of the world. Most have achieved very broad geographical distributions; 19 of 27 species are cosmopolitan or pantropical in distribution, and three of the remaining six species are distributed across at least two biogeographic regions (Table 4.1, Section 4). Equally, all species, with the exception of the Dysmicoccus finitmus, have very broad host ranges, encompassing host species in five to 124 plant families (median = 55 plant families, n = 25 species). Second, almost all are either well-known agricultural pests or recognized as significant quarantine threats (Miller et al. 2005; 2014). Third, the scale insect fauna on Christmas Island is largely shared with those reported for other islands across three ocean basins. Most of the scale insect species on Christmas Island are found repeatedly on other tropical or subtropical islands, even as far away as Bermuda in the Atlantic Ocean (>17,000 km). The only demonstrated pathway by which these species could consistently reach these isolated islands is through human- assisted dispersal. Soft scales (Coccidae), armored scales (), and (Pseudococcidae) are routinely intercepted in quarantine. Between 2007 and 2010 in US Ports of Entry (POEs) alone, Coccidae were intercepted 3,273 times, Diaspididae 40,000 times, and Pseudococcidae 25,064 times (Evans and Dooley 2013). Twenty-three of the 27 scale insect species on Christmas Island are entrained in worldwide human-associated dispersal pathways, as documented by interception records for scale insects at POEs in Asia (Republic of Korea - Suh et al. 2013) and North America (USA - Miller et al. 2014).

Endemic hemipterans on Christmas Island and host breadth of encyrtid parasitoids. Host breadth records of T. somervillei suggest that it will not attack endemic hemipterans on Christmas Island. We used the database to infer risk by considering whether any encyrtid parasitoid species have host ranges that include both scale insects and the families to which the endemic hemipterans belong. We obtained a list of known endemic insect species on Christmas Island (James and Milly 2006) and narrowed consideration of taxa on this list to the closest endemic relatives of lac scales on

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Christmas Island in the Order Hemiptera, the same order to which the known target species Tachardina aurantiaca belongs. These species comprise one true bug, a , a , a spittlebug, and three (Table 3.4). Two lines of evidence suggest the risk posed by T. somervillei to these species is extremely low. First, encyrtids are not common parasitoids of the families containing the endemics (Table 3.4). Indeed, 4 of the 7 families to which the endemic hemipterans belong have no known encyrtid parasitoids, and the remaining three are attacked by just 15 encyrtid parasitoid species, despite the great diversity (3735 species) in that group. Based on these data alone the probability of an encyrtid parasitoid attacking an endemic hemipteran on Christmas Island is extremely low.

Second, all of the endemic species occur in different suborders (either suborder Auchenorrhyncha or Heteroptera) to the yellow lac scale (suborder Sternorrhyncha)(see above and Fig. 2 in Cryan and Urban 2012). Thus, any potential for non-target impacts by T. somervillei on these endemic Hemiptera would require a host range that bridges this very substantial phylogenetic distance, as well as distinctive morphologies, life-histories and ecological attributes to its known host taxa in the Kerriidae.

B. Report on host specificity testing A host-specificity testing protocol was developed (Attachment 1) and then sought expert reviews were requested from five internationally known biological control practitioners in Australia, New Zealand, Switzerland, and the United States. Their opinions were sought on four main issues: the selection of test species, the use of no-choice tests over either choice or sequential no-choice tests, the design of tests, and the location and conditions under which the testing would be conducted. The reviewers' consensus was that testing be conducted under field conditions in the area of origin, to use replicated no-choice tests with comparable positive and negative controls, and to test species both closely related and more distantly related to the target Tachardina aurantiaca (Attachment 2). Despite the logistical challenges of conducting this kind of research in a foreign country, the host specificity testing proceeded according to this basic plan (Table 3.5). Study Site. Host specificity studies were conducted in Kuching (Sarawak, Malaysia) at a c. 24 ha site on the north side of the Sarawak River in the Kampung Boyan area (1o 33’ 42’’N, 110o 21’ 03’E). This area was chosen for several reasons: 1) the yellow lac scale, Tachardina aurantiaca, was found here at two different locations approx. 700 m apart in May 2012 on Acacia x A. auriculiformis; 2) T. somervillei, the natural enemy of choice as a biological control agent, was found parasitizing yellow lac scale at both of these locations but no other primary parasitoid was detected; 3) suitable yellow lac scale host plants were found throughout the area along with a variety of other plant species; and, 4) the area is not protected and, therefore, research permits

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were less complicated to acquire. The study site included several habitat types: settled area with gardens and backyards, managed parkland with higher diversity of plant species, moderately disturbed but unmanaged brush land with young trees, and secondary growth with older trees reaching 30 metres or more in height (Fig. 3.1A-D). Insecticide use was observed in 2012 but was limited to the settled area and targeted mosquitoes by fumigation in mosquito habitats. No pesticide use was observed during host specificity studies from August 2013 to March 2014. Tachardina aurantiaca and Tachardiaephagus somervillei at Kampung Boyan. Commencing June 2012, yellow lac scale crawlers were continuously moved within the study site from the two original locations to other host plants. Once adults had established, crawlers from these new locations were spread further through the study area until populations were established at 47 different locations, each with several lac scale aggregates. A "location" was defined as either a single host plant or group of host plants with overlapping canopies and the minimum distance between locations was 30 m. After 14 months (August 2013), no live yellow lac scale remained in any aggregates at 31 of the 47 locations. Although it was not possible to verify the source of mortality at these locations, parasitism by microhymenoptera was suspected since emergence holes were obvious in the dead lac scales. A subsequent collection of 30 mature female yellow lac scales at each of the remaining 16 locations with live scales showed evidence of parasitism by T. somervillei. No primary parasitoids other than T. somervillei were detected, suggesting that it alone was responsible for the elimination of yellow lac scale in all of the extinguished aggregates. Promuscidea unfasciativentris, a hyperparasitoid that attacks T. somervillei, was also present at the remaining aggregates with live yellow lac scale. Parasitoids dispersed naturally in the study area and were only moved among locations during the conduct of host specificity tests. These parasitoids were always contained in cages and were never reintroduced into the wild. Similarly, offspring that emerged during tests were never returned to the natural population. This avoided any artificial selection that might lower fitness of T. somervillei in the tests. The 16 locations with live yellow lac scale locations served as the source of parasitoids used in the tests. Yellow lac scale was propagated at these locations throughout the testing period to provide hosts for the parasitoid. When collecting parasitoids for testing, female yellow lac scales were examined at all 16 sites and females suspected to be parasitized and nearing parasitoid emergence were collected from as many sites as possible. Some parasitized females were always left intact to ensure the continuity of the local parasitoid population. Identifying parasitized females was relatively straightforward using a 20X hand lens. Healthy female Tachardina, or females that are parasitized with young larvae, are yellow on the sides and deep red or mostly red-brown on the top. In mature females nearing crawler release the bright red crawlers can sometimes be seen through the translucent test. In contrast, parasitized females with parasitoid pupae often show black areas under the test (see Fig. 1.3D in Section 1). Yellow lac scale females suspected of being parasitized were gently detached from their host plant and placed in a round plastic jar with a diameter and height of approximately 10 cm (Fig. 3.1E). A hole was drilled in the lid of the container just big enough to fit the opening of a 5-ml glass vial. The container was taped with black tape all around. Parasitoids emerging from the lac scales tended to move towards the light vial from the darker container, and then collected from the vial. The sex of the parasitoids was determined by examination of the antennae: females

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have club-like antennae shorter than the males’ antennae and males have longer antennae with whorls of setae (see Fig. 1.1 in Section 1). For each round of testing, parasitoids were randomly selected from a pool of individuals that emerged from lac scales collected at 3-5 locations within the study area. Only recently emerged parasitoids (<24 h) were used. Any remaining parasitoids were destroyed. This ensured that parasitoids used in the tests were the progeny of field- adpated – they had located their hosts without experimenter assistance, were exposed to intraspecific competition in the process of locating their hosts, and were sourced from several locations. Test Species. Selection of scale insect families for testing was based on phylogenetic distances inferred from genetic sequencing of nuclear 18S rRNA for 72 species in the Coccoidea (Fig. 3.2). Eight species were used in host specificity tests. In descending order of relatedness to the yellow lac scale, these included four soft scales (Coccidae), one diaspidid (Diaspididae) and three mealybug species (Pseudococcidae (Table 3.6 and Fig 3.3A-H). Three of the four species of Coccidae also occur on Christmas Island (Table 3.6). Some test species were sufficiently abundant at Kampung Boyan that enough naturally occurring aggregates could be located ( hesperidum, C. longulus, Paraputo near P. corbetti, and Chionaspis near C. broughae) for all tests. Other test species were less common (Milviscultulus mangiferae, Pseudococcus jackbeardsleyi, urbicola and Rastrococcus iceryoides) so additional aggregates were established by transferring crawlers to additional locations, but always to the same host plant species. All aggregates were inspected for natural parasitism prior to testing and when necessary, manipulated to ensure that only healthy, unparasitized females were enclosed in mesh bags. Parasitism in soft scales can usually be detected by visual inspection – compared to unparasitized females, parasitized females are noticeably darkened. As a further check, 30 individuals from three random aggregates for each species were collected and placed in glass vials for parasitoid emergence, if any, at the beginning of propagation (or before testing where propagation was not necessary). Initially, two test species (M. mangiferae and P. urbicola) were heavily parasitized and aggregates had to be culled and then newly established aggregates protected using mesh bags. Parasitism levels of were initially low and easily eliminated. Natural parasitism in the other test species did not occur over the study period. No-choice tests. The unit of replication in the no-choice tests was an aggregate of 25 mature female scale insects on a leafy branch, enclosed inside a fine mesh bag under field conditions (Fig. 3.1F). Five female and 5 male T. somervillei were liberated inside each bag (Day 0). The parasitoids were removed at Day 5 but the bag was left in place until Day 20, at which time the aggregate was cut from the plant and stored in plastic boxes until Day 35. Any emerging T. somervillei were counted. The aggregates were destroyed after Day 35 unless crawler emergence was detected; in that case the twig was returned to the host plant and fixed on it with a rubber band to allow crawler settlement and propagation of the yellow lac scale. The mesh bags excluded tending ants from test species during the period of exposure to the parasitoid. Tem replicates per species were used for each of the four coccid and three pseudococcid species, and five replicates for the diaspidid species. Replicates of Test Species were always located on the same species of host tree and usually spread across 2-7 trees. Replicates were established on different branches when more than one was used per tree. Each replicate involved a different batch of parasitoids, a different day of experimental set-up and a different group of test species individuals in a mesh bag, i.e. test species individuals were never reused even in the case of long-

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lived species. If any mortality was detected in a test group that was unlikely to be caused by the parasitoids (e.g. fungal disease), the replicate was discarded and replaced. Although conducted in the field, these no-choice tests were not completely open field tests. The tests were conducted within mesh bags, the parasitoids were confined, and interspecific competition was excluded. The pros and cons of this approach over testing under laboratory conditions in containment have been discussed elsewhere (see Discussion, below), but four of the five ad hoc expert reviewers supported this field approach, or preferred it over testing under laboratory conditions (Attachment 2). Finding Test Species replicates. Some test species were sufficiently abundant at Kampung Boyan that enough naturally occurring replicates could be found (Coccus hesperidium, C. longulus, Paraputo nr P. corbetti, and Chionaspis nr C. broughae). Other species were less common and additional aggregates were established by transferring crawlers to additional locations, but always to the same host species (Milviscultulus mangiferae, Pseudococcus jackbeardsleyi, Pulvinaria urbicola and Rastrococcus iceryoides). All aggregates were inspected for natural parasitism prior to testing and when necessary, manipulated to ensure that only healthy, unparasitized females were enclosed in mesh bags. Parasitism in soft scales is usually quite easy to detect by visual inspection – compared to unparasitized females, parasitized females are usually quite dark. As a further check, random samples of 30 individuals from 3 random aggregates in each species were also taken and placed in glass vials for parasitoid emergence, if any, at the beginning of propagation (or before testing where propagation was not necessary). Two test species, M. mangiferae and P. urbicola, were heavily parasitized in the beginning and the aggregates had to be culled and newly established aggregates had to be protected by mesh bags, while C. hesperidum showed low levels of parasitism at the commencement of testing but was easily cleaned. Natural parasitism in the other test species did not occur at the time of the study. Logistics dictated that not all replicates of a Test Species could be exposed to T. somervillei at the same time was seldom possible to run more than several replicates simultaneously due to the limited availability of aggregates of the test species, the availability of parasitoids, and the occasional loss of test replicates due to fungal disease. Therefore, the eventual total number of replicates for each Test Species was accumulated over several months of testing. Negative Controls. Negative controls, i.e. where test scale insect species are enclosed in mesh bags but without the parasitoid, were used to detect background mortality of scale insects in the absence of the parasitoid. As in no-choice tests, aggregates of 25 individuals were enclosed in mesh bags, but without parasitoids. For all test species, the same number of negative control replicates was used as in the test replicates. Negative controls were staggered in time to match each of the test replicates. Mortality in each negative control was evaluated seven days after the end of exposure to parasitoids in the corresponding no-choice tests. Positive Controls. Positive controls were used to determine the quality of the parasitoids being used against the test species – if T. somervillei failed to parasitize a test species, it could have been because the individuals used in the tests were somehow of inferior quality to “wild type”, rather than the test species being an unsuitable host per se. The validation of parasitoid quality is critical when parasitoids are reared in the laboratory because they may undergo laboratory selection, resulting in lower fitness. Laboratory conditions usually allow individuals of average or less than average fitness to reproduce (van Lenteren 2003). In our case, all parasitoids were

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collected from different locations in the wild, arguably obviating the need for these controls. However, to follow best practice, positive controls were used by retaining some of the reared T. somervillei individuals for testing against the natural host, Tachardina aurantiaca. Successful parasitization of these lac scales would demonstrate high parasitoid quality, and lessen the probability of false negatives in the test species. As in the no-choice tests, a positive control consisted of 25 unparasitized adult yellow lac scales, enclosed in a mesh bag to which 5 female and 5 males of T. somervillei were added. The yellow lac scales were exposed to parasitoids for 5 days, after which the parasitoids were removed from the bag. Yellow lac scales were left in the bags for a further 15 days, and then the branch was excised and placed in a plastic container to monitor parasitoid emergence over the next 10 days. As T. somervillei often superparasitizes its host (more than one progeny can emerge from a single host), the measure of parasitism used in the positive controls was the total number of emerging parasitoids rather than the percentage of parasitized hosts. Ideally, each no-choice and negative control replicate would have been accompanied by a positive control. Given the limited availability of aggregates of a given test species, variation in the availability of emerging parasitoids, and the occasional loss of test replicates due to fungal disease, it was not possible to complete host specificity testing for a scale insect species at one time. Therefore, the eventual total number of replicates for each test species was accumulated over several months. Parasitoids were produced in batches over this period that were then subdivided into lots of 10 individuals for use against test species replicates (Table 3.7). Thus, it was appropriate to test the quality of parasitoids at the batch level, rather than attempt to match every no-choice and negative control replicate with their own positive control. A randomly- selected subsample of each batch of parasitoids was reserved for testing against yellow lac scales, so that parasitoid quality was assessed regularly during the testing period. In all, 20 batches of parasitoids were produced over several months, but because of the vagaries of both parasitoid and yellow lac scale availability, 13 positive controls were conducted (Tables 3.7 and 3.8). Results of host specificity testing. In the no-choice tests, none of the eight test species were parasitized by T. somervillei. Not a single individual of T. somervillei emerged from any replicate aggregate in any species over several months of trials (Table 3.6). The absence of mortality in corresponding negative controls for each scale test species indicated that the mortality observed in the no-choice test replicates was attributable to parasitism by T. somervillei. Positive controls showed that all batches of T. somervillei used throughout the tests parasitized the yellow lac scale and near equivalent in terms of reproductive performance (Table 3.8). Emergence of adult parasitoids per replicate (18.7 ± 1.1 [SE], n = 13 and the per capita fecundity of female parasitoids (3.74 ± 0.22 progeny, n = 13) were similar in all 13 batches of T. somervillei released against aggregates of the natural (target) host the yellow lac scale and used against the test species yielded parasitoids (Table 3.8). Discussion. These field-based experiments and observations indicate that T. somervillei has an extremely narrow host range. First, during preliminary surveys at the field site it never successfully attacked any of the other scale insects used in this study. Second, the parasitoid did not successfully parasitize any of the tested species during no-choice tests, despite being put under pressure to do so by the lack of a suitable alternative host (i.e. the yellow lac scale). Further, the tested scale insect species occur in the native range of the parasitoid and often

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shared habitat with the yellow lac scale. , for example, occurred on the same host plant (Acacia mangium x A. auriculiformis) as the yellow lac scale, and often in mixed aggregates. Yet, C. longulus was never parasitized by T. somervillei under natural conditions. We used outdoor mesh bags to carry out the tests on host plants on which the test species were originally found. Sands and Van Driesche (2003) emphasize that such test conditions have some advantages over laboratory tests because the full range of the parasitoid’s behaviors that lead to host acceptance, feeding, and oviposition can occur in the natural environment of the parasitoid’s habitat. The use of wild-collected parasitoids in the tests also eliminated the chance of reduced parasitoid fitness, which might otherwise have been the case with laboratory-reared parasitoids. The possible negative ‘arena effects’ that could influence parasitoid behavior and subsequently parasitism (Sands and Van Driesche 2003) were ruled out by the positive controls, because the target hosts were exposed to the parasitoid in the enclosures, as were the non-target test species. Without exception, the positive controls yielded parasitoid emergence suggesting that it was capable of high levels of parasitism in the mesh bags, and the parasitoid batches collected from the wild were of consistently high quality. A reasonable inference then is that the parasitoids from the untested batches (Table 3.8) were of equal quality and that the replicates of test species using these batches are still valid. Soft scale insects (Coccidae), the family most closely related to lac scales (Kerriidae), were most represented in the test species list. The database analysis of host records (Section 3A) showed that most encyrtid primary parasitoid species (32/40 species, 80%) were restricted to host species in the Kerriidae. Of the few remaining parasitoid species, their most probable alternative hosts were soft scales and no reliable host records were found outside of the Coccoidea. Mealybugs (Pseudococcidae) represent a farther phylogenetic distance than soft scales and the lack of parasitism in the mealybug test species in field tests was expected, given the failure of T. somervillei to parasitize the more closely related coccid and diaspidid species. The results of the field study strongly suggest that T. somervillei has a narrow host range limited to species in the family Kerriidae (lac scales), consistent with inferences from the taxonomic literature and historical records (see Section 3A). These data suggest further that no other scale insect species on Christmas Island are at risk from the introduction of T. somervillei, with the possible exception of the lobate lac scale, Paratachardina pseudolobata (Kerriidae). However, this species is not native, an invasive environmental pest in its own right (Howard et al. 2007), and therefore not of conservation significance. The endemic hemipteran taxa on Christmas Island fall far outside the narrow host range of T. somervillei, all occurring in two suborders (Auchenorrhyncha or Heteroptera) that are phylogenetically distant from the Sternorrhyncha, the suborder to which the target species belongs (see Section 3A above). All available evidence suggests that the host range of T. somervillei is substantially too narrow to bridge such phylogenetic distance, and that the risk posed to these species by the introduction of T. somervillei to Christmas Island is extremely low.

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TABLES and FIGURES

Table 3.1. Records of known host families and genera1 for the primary parasitoid Tachardiaephagus (Encyrtidae). The proposed biological control agent, Tachardiaephagus somervillei, is in bold. As a genus, Tachardiaephagus has an extremely broad geographic range. With the exception of one host record in Africa reported as erroneous by Prinsloo (1977), all Tachardiaephagus species appear to be family specialists and restricted to the Kerriidae. For host genera, number of species recorded as hosts is in parentheses. Based on Noyes (2012, Universal Chalcidoidea Database, http://www.nhm.ac.uk/research- curation/research/projects/chalcidoids/ database/), except for additional records for T. somervillei and T. sarawakensis (Hayat et al. 2010; Green et al. 2013; R.W. Pemberton, pers. comm.).

Parasitoid species Distribution Recorded hosts (all Kerriidae)

Tachardiaephagus India, Malaysia, Thailand Kerria spp. (4)1 somervillei Tachardina aurantiaca Tachardina sp.2

T. sarawakensis Sarawak (East Malaysia) Tachardina aurantiaca

T. tachardiae Brunei, China, India, , Kerria spp. (8) Malaysia, Sri Lanka, Taiwan, Paratachardina lobata3 Vietnam, Azerbaijan (= P. silvestri)

T. similis Afrotropical, South Africa Tachardina sp. (1)

T. absonus Afrotropical, South Africa Tachardina spp. (2)

T. communis Afrotropical, South Africa Tachardina spp. (5)

T. gracilis Afrotropical, South Africa Tachardina sp. (1)

1 Ben-Dov et al. (2012) indicate that the kerriid genus Laccifer is a synonym for the genus Kerria. Therefore, we have combined records for Laccifer spp. in Noyes (2012) with records for Kerria. 2Probably T. aurantiaca, since it is the only Tachardina species known in Asia. 3Noyes (2012) lists P. lobata as a host for T. somervillei. However, this is based on an incorrect reading of Pemberton 2003; the author of that paper states that while T. somervillei has not been recorded from P. lobata, it was worth testing T. somervillei against P. lobata because the congeric species T. tachardiae had been recorded to parasitize it.

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Table 3.2. Known host range of primary parasitioid species in the 16 genera of encyrtids (Chalcoididea: Encyrtidae) known to attack members of the lac scale family Kerriidae (the family of the target insect Tachardina aurantiaca). Tachardiaephagus is in bold. Data from the Universal Chalcidoidea Database - Noyes 2013). Phylogenetic distance in host range from the Kerriidae increases from left to right, i.e from the Coccidae to 'other neococcids' to 'archeococcids' (see Gullan and Cook 2007).

Known host range

Encyrtid genus No. Kerriidae Kerriidae + Kerriidae + Kerriidae + species only Coccidae 'neococcids' Coccidae only +'archeococcids'

Adencyrtus 3 2/3 1/3 - -

Ageniaspis 1 1/1 - - -

Ammonoencyrtus 1 - 1/1 - -

Clausenia 1 1/1 - - -

Coccidaphycus 1 - - - 1/1

Coccopilatus 1 1/1 - - -

Erencyrtus 6 6/6 - - -

Laccacida 1 1/1 - - -

Lakshaphagus 1 1/1 - - -

Metaphycus 7 3/7 2/7 2/7 -

Microterys 2 1/2 1/2 - -

Ooencyrtus 3 3/3 - - -

Ruandella 1 1/1 - - - Tachardiaephagus 7 7/7 - - - Tachardiobius 3 3/3 - - - Tyndarichus 1 1/1 - - -

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Table 3.3. Fourteen of the 18 endemic plant species on Christmas Island searched for endemic scale insects (http://laptop.deh.gov.au/parks/christmas/nature/flora). N is the number of individuals or clusters of plants examined in each of three years. See Green et al. (2013) for search locations.

Species (Family) Growth form Habitat N

Common endemics Hoya aldrichii (Asclepiadaceae) vine Plateau 30 Arenga listeri (Arecaceae) tree palm Plateau/Terrace 30 Brachypeza archytas (Orchidaceae) epiphyte Terraces 30 Pandanus christmatensis shrub/tree Terraces 30 (Pandanaceae) Pandanus elatus (Pandanaceae) tree Plateau/Terrace 30

Rare endemics Asystasia alba (Acanthaceae) herb Coastal/Terrace 5 Asplenium listeri (Aspleniaceae) fern Terrace 4 Abutilon listeri (Malvaceae) shrub Coastal/Terrace 12 Flickingeria nativitatis (Orchidaceae) epiphyte Plateau 25 Phreatia listeri (Orchidaceae) epiphyte Plateau 12 Ischaemum nativitatis (Poaceae) grass Coastal 4 Colubrina pedunculata (Rhamnaceae) shrub Terrace 30 Grewia insularis (Tiliaceae) shrub/tree Terrace 25 Dendrocnide peltata var. murrayana tree Terrace 8 (Urticaceae) Rare endemics not searched Dicliptera maclearii (Acanthaceae) herb Coastal/Terrace -- Peperomia rossi (Piperaceae)1 herb Plateau -- Zehneria rossi (Cucurbitaceae) vine Coastal/Terrace -- Zeuxine exilis (Orchidaceae)2 herb Plateau -- 1Probably extinct 2Thought extinct until recently (see Green et al. 2010)

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Table 3.4. Endemic hemipteran species known from Christmas Island and primary parasitoids (superfamily Chalcidoidea: family Encyrtidae) associated with the families represented by the endemic species. The data were extracted from the Universal Chalcidoidea Database (Noyes 2012). The families Nogodonidae and have no associated chalcidoid primary parasitoids and therefore endemic species in these families on Christmas Island can most likely be excluded from all further consideration. Cicadellidae and have the highest diversity of chalcidoid primary parasitoids but have orders of magnitude lower diversity of encyrtid primary parasitoids. These data suggest that the encyrtid primary parasitoids of families with endemic species on Christmas Island appear to not have host range overlap with taxa where the target lac scale is included and the host range separation is at the suborder level. This suggests very distant phylogenetic separation. During the database analysis, only records with species-level chalcidoid identification were used. N/A indicates not applicable.

Endemic species Family No. chalcidoid No. encyrtid 1o Suborder/Family host associates parasitoid range of encyrtids of family species of family parasitizing family

Xestocephalus Cicadellidae 627 6 Auchenorrhyncha izzardi1 (Cicadellidae) Oxypleura Cicadidae 35 0 N/A calypso

Clovia eximia 71 4 Auchenorrhyncha (Cercopidae- ) Ugyops aristella Delphacidae 248 5 Auchenorrhyncha (Delphacidae- Cicadellidae)

Varcia 0 N/A N/A flavicostalis Salona oceanica

Leptocoris Rhopalidae 0 N/A N/A subrufescens2

1 Xestocephalus izzardi is also reported from Palau in the western Pacific Ocean (Linnavuori 1975). Its status as an endemic on Christmas Island is questionable. 2 Leptocoris subrufescens on Christmas Island has been classified to subspecies status (L. subrufescens subrufescens). Another subspecies (L. s. flava) is described from Yap, western Pacific Ocean (Göllner- Scheiding 1980). More research is needed to resolve the taxonomic status of these two subspecies of L. subrufescens.

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Table 3.5. Comparison of key features of the Host Testing Protocol that was peer-reviewed by expert biological control practitioners, and the actual methods used for the Testing.

Key Feature of Externally Original Parameters Eventual Outcome Reviewed Protocol

Number of test species 10 to 15, but fewer if high specificity is found initially 8 species tested Relatedness of test species Focus on neococcid taxa including the Kerriidae, the family to 4 Coccidae species tested to the target species, which the target host belongs (see Gullan and Cook 2007). No Kerriidae other than Tachardina aurantiaca tested Tachardina aurantiaca Considering the phylogenetic relationships of scale insect families, aim to test more than one species from the family Coccidae Inclusion of phylogenetic Species in the family Diaspididae will also be considered as a less 1 Diaspididae species tested ‘outgroups’ closely related group of scale insects. Early in the host testing 3 Pseudococcidae species tested process, an ‘out-group’ (test species phylogenetically more distant) will also be used, most likely selected from the family Pseudococcidae

Approach to testing No-choice tests preferred over choice tests or sequential no-choice No-choice tests used, each replicate paired with a tests. Tests accompanied by both positive controls (test parasitoid negative control. Positive controls conducted on against known host to confirm their quality) and negative controls ‘batches’ of parasitoids produced for tests, rather than (test species enclosed without parasitoids to determine paired with test and negative control replicates background mortality) Location of testing Field trials in area of origin preferred over testing in containment All field trials were conducted within the area of origin at the release location (Christmas Island), or testing in at a site in Kuching (Sarawak) containment on mainland Australia

Replication In no-choice tests, 10 trials per test species, with 50 individuals of There were 10 replicates for 7 test species, and 5 the test species exposed to 10 female and 10 male parasitoids. replicates for the other test species. There were 25 Hosts and parasitoids enclosed on branches by mesh bags individuals of the test species per trial, exposed to 5 female and 5 male parasitoids

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Table 3.6. Test species, their host plants, replication and experimental outcomes for the host-specificity testing conducted at Kampung Boyan near Kuching (Sarawak, Malaysia) in 2013-2014. Nreps is the number of independent replicates undertaken for each test species (see Table 3.7). Ninsects/rep is the number of individuals of a given test species used in each replicate. *species occurs on Christmas Island.

Test Species (Family) Host Plant Species (Family) Nreps Ninsects/rep No. emerging Progeny per parasitoids/rep female parasitoid

Coccus hesperidum (Coccidae)* Ficus sp. (Moraceae) 10 25 0 ± 0 0 ± 0

Coccus longulus (Coccidae) Acacia sp. (Mimosaceae) 10 25 0 ± 0 0 ± 0

Milviscutulus mangiferae (Coccidae)* Morinda citrifolia (Rubiaceae) 10 25 0 ± 0 0 ± 0

Pulvinaria urbicola (Coccidae)* Pisonia grandis (Nyctaginaceae) 10 25 0 ± 0 0 ± 0

Chionaspis near C. broughae (Diaspididae) Mangifera sp. (Anacardiaceae) 5 25 0 ± 0 0 ± 0

Paraputo near P. corbetti (Pseudococcidae) Mangifera sp. (Anacardiaceae) 10 25 0 ± 0 0 ± 0

Pseudococcus jackbeardsleyi (Pseudococcidae) ?Aglaonema sp. (Araceae) 10 25 0 ± 0 0 ± 0

Rastrococcus iceryoides (Pseudococcidae) Croton sp. (Euphorbiaceae) 10 25 0 ± 0 0 ± 0

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Table 3.7. The number replicates (solid circles in each column) for each test species, the batch of T. somervillei used for each test replicate, and which of those batches were quality-tested in Positive Control trials (see Table 3.8). For test species, Ch = Coccus hesperidum, Cl = Coccus longulus, Mm = Milviscutulus mangiferae, Pu = Pulvinaria urbicola, Cb = Chionaspis near C. broughae, Pc = Paraputo near P. corbetti, Pj = Pseudococcus jackbeardsleyi, Ri = Rastrococcus iceryoides.

Test Species

Parasitoid Ch Cl Mm Pu Cb Pc Pj Ri Positive control batch 1 • • • • Yes

2 • • • No

3 • • • • Yes

4 • • • • • Yes

5 • • • • • • • Yes

6 • • • • • • • Yes

7 • • • • • • • No

8 • • • • • • • Yes

9 • • • • • • • Yes

10 • • • • • • • No

11 • • • Yes

12 • • • • Yes

13 • • Yes

14 • • No

15 • No

16 • Yes

17 • No

18 • Yes

19 • No

20 • Yes

Total Test 10 10 10 10 10 10 10 5 Replicates

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Table 3.8. Performance of batches of T. somervillei used as the positive controls in host specificity testing. A total of 20 batches of parasitoids were produced for use in the host specificity testing. T. somervillei from 13 batches were tested against the natural (target) host, Tachardina aurantiaca under field conditions over several months of trials at Kampung Boyan near Kuching (Sarawak, Malaysia) in 2013-2014.

Batch No. ♀ T. No. ♀ T. No. Emerging Parasitoids Progeny per aurantiaca somervillei parasitoids per scale ♀ parasitoid (host) (agent)

1 25 5 17 0.68 3.4

3 25 5 12 0.48 2.4

4 25 5 14 0.56 2.8

5 25 5 21 0.84 4.2

6 25 5 18 0.72 3.6

8 25 5 16 0.64 3.2

9 25 5 19 0.76 3.8

11 25 5 22 0.88 4.4

12 25 5 20 0.80 4.0

13 25 5 26 1.04 5.2

16 25 5 24 0.96 4.8

18 25 5 15 0.60 3.0

20 25 5 19 0.76 3.8

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Figure 3.1 A – D. The variety of vegetation types in which the host specificity tests were conducted at Kampung Boyan near Kuching (Sarawak, Malaysia). The vegetation varied from managed parkland to secondary rainforest. E. The device used to capture individuals of the agent Tachardiaephagus somervillei as they emerged from detached individuals of the host Tachardina aurantiaca. The large plastic container was covered in black tape, and the parasitoids tended to fly up into the small glass vial fitted to the larger plastic lid, where they could be easily collected. F. A replicate used for host specificity testing. The mesh bag enclosed 25 unparasitized individuals of the test species, together with 5 female and 5 male T. somervillei. The same mesh bags were used for negative controls (test species without exposure to parasitoids) and for positive controls (T. aurantiaca enclosed with T. somervillei). Photos: G. Neumann.

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Figure 3.3. The eight species used in the host specify testing, arranged in descending order of relatedness to the target species Tachardina aurantiaca (Kerriidae); Coccidae – A. Coccus hesperidum Linnaeus, B. Coccus longulus (Douglas), C. Milviscutulus mangiferae (Green), D. Pulvinaria urbicola Cockerell. Diaspididae – E. Chionaspis near C. broughae Williams & Watson. Pseudoccidae – F. Paraputo near P. corbetti (Takahashi), G. Pseudococcus jackbeardsleyi Gimpel and Miller, H. Rastrococcus iceryoides (Green). Photos: G. Neumann except B Coccus longulus by Ian Stocks courtesy of http://bugguide.net/node/view/487910

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SECTION 4

An assessment of the possible direct and consequential impacts to the environment if soft scale insects, such as Pulvinaria urbicola, were to fill the niche left by reducing yellow lac scale numbers. The assessment should discuss and describe any proposed biological control of the soft scale insects. [in addition, we assess the potential for yellow crazy ants to use a different source of food to maintain supercolonies if the abundance of scale insects is successfully reduced through the biological control program]

[Some of these issues were addressed in the initial submission (ER Part 2.2.2 Develop a biological control program for several species of soft scales, pp. 13-14; RA Part A, Risk Ref/ID5, p. 24; RA Part B, Risk Ref/ ID5, p. 27] A. Potential expansion of the feeding niche of soft scale insects following suppression of population densities of T. aurantiaca by Tachardiaephagus somervillei.

Tachardina aurantiaca is one of four non-native, honeydew-producing scale insect species that are common in yellow crazy ant supercolonies on Christmas Island (Table 4.1). Although soft scales, principally Coccus spp. and Saisettia spp., have been recorded from most of the main host plants of Tachardina in supercolonies (e.g. Inocarpus fagifer, Terminalia catappa, Tristiropsis acutangula, Ficus microcarpa and Milletia pinnata), they occur at much lower densities than on plant species that are not major hosts of T. aurantiaca (e.g. Planchonella nitida, Syzygium nervosum, Dysoxylum gaudichaudianum, and Clayoxylon indicum) (O’Dowd et al. 2003, Abbott 2004). The feeding niche of these soft scale insects is distinctive to that of Tachardina aurantiaca. Coccus spp. and Saisettia spp. (Coccidae) primarily feed and develop on leaf tissues whereas T. aurantiaca consistently feeds and develops on stems < 1 cm in diameter. This segregation is maintained independently of the presence or absence of T. aurantiaca, strongly suggesting that direct competition for feeding sites is unlikely to generate patterns of within-host species or between- host species segregation. A more likely explanation for segregation of feeding niches is related to the morphology of the mouthparts of these scale insects relative to the location of plant phloem tissue, nutritional quality of phloem contents, and plant secondary chemistry. If so, suppression of Tachardina following the importation and release of Tachardiaephagus should not lead to population increases in soft scale insects, compensatory production of honeydew, and the maintenance of yellow crazy ant supercolonies. Nevertheless, concurrent plans to redistribute two existing parasitoids (Coccophagus ceroplastae and Encyrtus infelix) of soft scale insects in rainforest should reduce soft scale densities, independent of the abundance of Tachardina aurantiaca (see Section 4B below).

B. Biological control of soft scale insects

Although Tachardina is strongly implicated as the main contributor to the honeydew economy of yellow crazy ants in supercolonies, there is considerable site-to-site variation in its likely contribution, from 46-86% (Green et al. 2013). While Tachardiaephagus somervillei should provide a high level of biological control over populations of Tachardina aurantiaca, it is not certain that targeting this species alone would provide consistent indirect control for the yellow crazy ant in all supercolonies, especially where its contribution to the honeydew budget is lowest. Based on information presented in Green et al. (2013), the Crazy Ant Scientific Advisory Panel (see Section 5) advised Parks Australia that the program on Christmas Island be expanded to include agents for the control of these coccid soft scales. Complementary use of agents against both Tachardina and the soft scales could provide a high and consistent level of control for the entire assemblage of honeydew-producing scale insects, and thus increase the likelihood of consistent, sustainable suppression of yellow crazy ant supercolonies.

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Three parasitoid species that already attack honeydew-producing soft scale insects on Christmas Island are Coccophagus ceroplastae and C. longifasciatus (Hymenoptera: Aphelinidae), and Encyrtus infelix (Hymenoptera: Encyrtidae). Both C. ceroplastae and E. infelix have been cultured and used with success elsewhere in the biological control of soft scale insects in nature reserves, agriculture and horticulture. For example, C. ceroplastae was introduced and released to manage Pulvinaria urbicola in the Coringa-Herald National Nature Reserve in the Coral Sea Islands Territory (Smith et al. 2004). On Christmas Island, these parasitoid species were discovered as part of the research and development program for biological control of Tachardina aurantiaca (Green et al. 2013) and attack both Coccus and species that are abundant in some yellow crazy ant supercolonies (Table 4.2). C. ceroplastae, C. longifasciatus and E. infelix were almost certainly introduced inadvertently to the island with importation of plant material with host scale insects during human settlement and prior to effective quarantine measures. The host ranges of all three parasitoids are both relatively well known, and include honeydew-producing coccid species in rainforest on Christmas Island (e.g. Coccus hesperidium, nigra, Saissetia spp., Pulvinaria spp. - Noyes 2014). None have ever been recorded from kerriid lac scale species (Noyes 2012) or parasitizing Tachardina aurantiaca on Christmas Island, despite their co-occurrence in some areas. These observations indicate that none of these agents use the target species T. aurantiaca as a host and would not affect the establishment and efficacy of T. somervillei against T. aurantiaca through competition for the same host. The use and redistribution of these parasitoids, already well established on Christmas Island, obviates the difficult issues of foreign exploration and host-specificity testing. Experience with the efficacy of these agents in dealing with the outbreak of Pulvinaria urbicola on Pisonia spp. on Christmas Island (Neumann et al. 2011) suggests that the current lack of control of soft scales in yellow crazy ant supercolonies is a result of dispersal limitation of their parasitoids and C. ceroplastae has already been distributed at sites with Pisonia grandis to control the soft scale Pulvinaria urbicola (Neumann et al. 2011). Dispersal limitation will be overcome by releasing one or more of these parasitoid species at multiple sites and at multiple times across the island. Although initial rearing and release will focus on T. somervillei, preparations are also being made for the mass rearing of C. ceroplastae and E. infelix on Christmas Island. The two programs are integrated: the same scientific staff oversee both programs, the same natural resource agency staff are propagating plants and will assist in rearing hosts and parasitoids, and both programs are funded as a single entity by the Department of Environment.

C. The potential for yellow crazy ants to utilize a different source of food to maintain supercolonies if the abundance of mutualistic scale insects is successfully reduced through the biological control program

If honeydew from scale insects becomes scarce as a result of the biological control program against both yellow lac scales and soft scales, could yellow crazy ants utilize a different source of food to maintain their supercolonies?

Yellow crazy ants use a range of food sources – broadly speaking they obtain carbohydrate from plants, and protein from . Ants harvest plant carbohydrates either directly from the plant (nectar secreted from flowers or from glands on leaves), or indirectly as honeydew from scale insects. The protein is derived largely from animals they kill – mostly small invertebrates, but also land crabs. There are several lines of evidence to indicate that supercolonies can only form where the ants have access to large amounts of honeydew, which they themselves create by ‘farming’ the scale insects, and that nectar and animal prey alone or in combination are insufficient to sustain supercolonies (See Attachment 3, Research projects 1a- 1d for empirical evidence supporting the four points outlined below)

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First, supercolony densities of yellow crazy ants invariably co-occur with outbreak densities of honeydew- producing scale insects. Supercolonies have never been observed to form in the absence of high densities of scale insects – that is, yellow crazy ants have never reached high densities simply by harvesting floral or extra-floral nectar, and/or killing invertebrates.

Second, dietary analyses using stable isotopes indicate that yellow crazy ants are largely herbivorous when they occur at supercolony densities. This is consistent with the idea that honeydew from scale insects – a plant product – sustains them at high density. In these analyses, crazy ants were never sustained at high densities by a largely carnivorous diet, indicating that if they are denied access to honeydew through the biocontrol program for scale insects, the ants will not be able to move onto a protein-rich food source in order sustain their high densities.

Third, a laboratory experiment demonstrated that colonies of captive yellow crazy ants performed best (high queen productivity, low worker mortality, high worker foraging tempo, high worker aggression towards other species of ants) only when fed high amounts of sugar in their diet. Colonies fed low amounts of sugar performed significantly less well, despite all colonies being fed excess protein. This experiment suggests that it is sugar, not protein, that is key to supercolony formation.

Fourth, a field experiment showed that supercolonies decline precipitously when denied access to honeydew-secreting scale insects. In supercolonies, most scale insects occur in the canopies of large trees. Bands placed at breast height on tree trunks prevented ants from climbing trees to reach the scale insects, and ant densities dropped five-fold within four weeks, compared with pre-treatment levels. In the days after banding, less than 1% of the ants descending the trees were carrying prey items, but most had obviously swollen, translucent abdomens indicating they had been foraging mostly for honeydew. This experiment showed not only that biological control of scale insects is likely to be effective in reducing ant densities, but that the ants were unable to utilise an alternative, ground-based food source to sustain their numbers.

In summary, following the release and population build-up and spread of T. somervillei on Christmas Island, both its abundance and that of its host, Tachardina aurantiaca should follow classic parasitoid-host dynamics, and both should decline to low population densities similar to the situation observed in the native distribution of both organisms in Southeast Asia. All the available evidence indicates that yellow crazy ants will not be able to sustain high-density supercolonies in the near-absence of their mutualistic scale insects.

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TABLES and FIGURES

Table 4.1. Scale insects of Christmas Island. It is highly probable that all of these species, with broad host plant ranges and geographic distributions, are exotic to Christmas Island and introduced following human settlement. The target species for biological control, Tachardina aurantiaca, is in bold. Honeydew-producing scale insects (in bold) are commonly tended by the yellow crazy ant in supercolonies. Families are arranged in increasing phylogenetic distance from the Kerriidae based on Gullan and Cook (2007) and Ross et al. (2012). All scale insect taxa are 'neococcids' except for Nipponorthezinella guadacanalia and Icerya purchasi ('archeococcids'). Taxonomy, most common names, distributions (AFR = Afrotropical; AUS = Australasian; COS = Cosmopolitan; NEA = Nearctic; NEO = Neotropical; ORI = Oriental; PAL = Palaearctic; PAN = Pantropical) , and most host ranges (number of plant families) from Ben-Dov et al. (2014).

Honeydew Family and Species1 Common Name Distribution Host range Producer

Kerriidae (lac scales) Paratachardina pseudolobata Lobate lac scale ORI,NEA, 66 yes2 (Kondo & Gullan) NEO Tachardina aurantiaca (Cockerell) Yellow lac scale ORI 34 yes Coccidae (soft scales) (Fabricius) Indian wax scale COS 52 yes C. destructor Newstead Soft wax scale AFR,AUS, 28 yes ORI Coccus celatus De Lotto Green coffee scale AFR,AUS, 10 yes ORI C. hesperidum Linnaeus Brown soft scale COS 124 yes Milviscutulus mangiferae (Green) Mango shield scale COS 40 yes (Nietner) Nigra scale COS 93 yes Pulvinaria urbicola Cockerell Urbicola soft scale COS 43 yes P. psidii Maskell Green shield scale COS 63 yes (Walker) Hemispherical scale PAN 111 yes S. oleae (Olivier) Black scale COS 78 yes Diaspididae (armoured scales) Aspidiotus destructor Signoret Coconut scale COS 68 no Hemiberlesia palmae (Cockerell) Tropical palm scale COS 55 no longirostris (Signoret) Black thread scale COS 57 no Lepidosaphes beckii (Newman) Citrus mussel scale COS 42 no Lindingaspis tingi McKenzie -- ORI 5 no Pseudaulacaspis pentagona White peach scale COS 88 no (Targioni Tozzetti) citri (Comstock) White louse scale COS 14 no (ornate pit scales) Cerococcus indicus (Maskell) Spiny brown coccid COS 11 yes Pseudococcidae (mealybugs) Dysmicoccus finitimus Williams Asian coconut mealybug AUS,ORI 1 yes Ferrisia virgata (Cockerell) Striped mealybug COS 83 yes Nipaecoccus viridis (Newstead) Spherical mealybug COS 44 yes Pseudococcus longispinus Long-tailed mealybug COS 90 yes (Targioni Tozzetti) Orthezidae (ensign scales) Nipponorthezinella guadacanalia -- ORI,AUS ?3 ? (Morrison) AFR (giant scales) Icerya purchasi Maskell Cottony cushion scale COS 69 yes 1Records from Campbell (1968), CSIRO (1990), O'Dowd et al. (2003), Bellis et al. (2004), Abbott (2004), Woods and Steiner (2012) and Neumann et al. (unpubl. results). 2Paratachardina pseudolobata produces honeydew but it is ejected and appears unattractive to ants (Howard et al. 2010). 3Nipponorthezinella guadacanalia has been collected from the soil and may be a root-feeding species. Its host plant range is not known.

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Table 4.2. Scale insects species on Christmas Island that are known hosts of three chalcidoid parasitoids (Noyes 2014) introduced to the island since human settlement. All scale insect species listed, except the diaspidid Aspidiotus destructor, are honeydew producers. Scale insect species in bold are those that are commonly tended by yellow crazy ants in supercolonies (see Table 4.1).

Coccophagus ceroplastae Coccophagus longifasciatus Encyrtus infelix (Aphelinidae) (Aphelinidae) (Encyrtidae)

Coccidae Coccidae Coccidae Coccus hesperidum Coccus hesperidum Ceroplastes destructor Parasaissetia nigra Parasaissetia nigra Coccus hesperidum Pulvinaria psidii Parasaissetia nigra Pulvinaria urbicola Pulvinaria urbicola Saissetia coffeae Saissetia coffeae Saissetia oleae Saissetia oleae Diaspididae Aspidiotus destructor Pseudococcidae Nipaecoccus viridis

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SECTION 5 Further detail regarding the role and membership of the Crazy Ant Scientific Advisory Panel, such as Terms of Reference and the minutes of the meeting of 12 December 2012, when the Panel considered this proposed action.

Following a report submitted to the Department of Environment in 1999 (O'Dowd et al. 1999), the then Australian Nature Conservation Agency (ANCA) brought together a committee of experts on invasive ants, conservation biology, and natural resource management (The Christmas Island Crazy Ant Steering Committee - CICASC) to advise Parks Australia on the management of the yellow crazy ant Anoplolepis gracilipes on Christmas Island. In July 2008, following federal government approval and a line item in the budget, funding the first four years of a ten-year Crazy Ant Management Strategy, the committee was reconstituted as the Crazy Ant Scientific Advisory Panel (CASAP) with a new Terms of Reference (Attachment 4), which includes a list of members and observers. The final minutes of the teleconference meeting on 12 December 2012, when CASAP considered this proposed action, are also attached (Attachment 5).

See Attachment 4. Terms of Reference for the Crazy Ant Scientific Advisory Panel.

See Attachment 5. Final minutes of the CASAP teleconference on 12 December 2012.

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SECTION 6 Proposed methodology for selection of release sites, monitoring of potential impacts and adaptive management.

[Some of these issues were addressed in the original Environmental Referral (EF Part 2.1 Approval, Importation & Rearing, and Release & Monitoring of Tachardiaephagus, pp. 12- 13; RA Part A Identifying and Analysing Project Management Risks a. Risk Ref/ID1, p. 21-22, b. Risk Ref/ID2, p. 22, c. Risk Ref/ID3, p. 23, d. Risk Ref/ID4, p. 23-24, e. Risk Ref/ID5, p. 24; and, RA Part B Risk Register & Mitigation - Risk ref/IDs 1-5, pp. 25-27; Location addressed in original submission: ER Part 2.1 Release & Monitoring, p. 13]

A. Mass rearing and field release A dedicated screen house production facility is being built by Parks Australia on Christmas Island to mass-rear Tachardiaephagus for field release. As in Malaysia, the maintenance of captive parasitoid populations depends on the production and maintenance of optimal host life stages of Tachardina on suitable host plants. The best locally available host plant species for Tachardina are Inocarpus fagifer and Milletia pinnata, but Tristiropisis actutangula, Ficus microcarpa and Terminalia catappa are also being produced. The rearing of the parasitoid will occur in two stages. First, potted host plants will be transferred into one half of the screenhouse where they will be inoculated with Tachardina. Once populations have built up, plants will be relocated into the other half of the screen house where the Tachardina populations will be exposed to Tachardiaephagus. Careful consideration has been given to the design of the screen house to enable the internal transfer of plants between the two sides of the facility, while containing the parasitoid to the second half. Tachardina and Tachardiaephagus may be difficult to mass-rear due to the relatively long life cycle of Tachardina (from crawler stage to reproductive female is 80-100 days, but generations overlap) and the need for fresh host plants (that may or may not be reused) on a regular basis. Depending on the difficulty of rearing Tachardiaephagus, their availability for releases maybe limited at any given time. As mass-rearing methods are adapted and production increases on Christmas Island, the goal will be to provide the biological control agent for releases in all areas as needed. The population of Tachardiaephagus will require careful monitoring and maintenance to minimize any selection of shade house-adapted insects that perform poorly under field conditions. It may be necessary to replenish the genetic diversity of the captive population through the subsequent introduction of more insects from the founder population in Malaysia, or, more likely, from the field on Christmas Island once the initial releases have been conducted. Population renewal will also counter the inherent susceptibility of microhymenoptera to the loss of population heterozygosity. Sex determination in microhymenoptera is usually haplodiploid – males are haploid, females diploid, and heterozygosity at a multi-allelic sex-determining locus is required for femaleness. Inbreeding can lead to a preponderance of homozygous diploids that will either be sterile males, or can experience a very high rate of mortality. B. Field release of Tachardiaephagus. The goal in releasing biological control agents is to generate sufficient ‘propagule pressure’ (e.g., the size of each release, the frequency of

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releases, and the number and spatial arrangement of release sites) to enable their successful establishment. Increasing propagule pressure can enhance the likelihood of establishment by diminishing the role of chance (i.e., both demographic and environmental stochasticity), and potentially increase the rate of spread from release sites (Simberloff 2009). Initial timing, number of individuals released, and the frequency of releases of both agents will depend on (a) the capacity and sustainability of mass rearing, (b) knowledge of the biology of Tachardiaephagus, especially in relation to its host, Tachardina; and, (c) the attributes of release sites. Criteria for choosing suitable primary release sites will include: (a) positive evidence of host scale infestation; (b) relatively high percentage of host plants of Tachardina or soft scale in the overstorey and understorey; (c) occurrence of a high-density supercolony of yellow crazy ants or at least their presence; and, (d) site not subjected to current pesticide exposure (contact or systemic) or residues that could compromise establishment of the agents. Some criteria (e.g., c and d) can be gleaned from the biennial Islandwide Survey (Green and O'Dowd 2009) and will be followed up by more detailed site assessments to determine (a) and (b). A specified number of adult T.somervillei will be released at each selected site. Fipronil, the intoxicant in Antoff®, the bait currently used to control yellow crazy ant supercolonies on Christmas Island, is known to affect the longevity, fecundity, and behaviour of some parasitoids. For example, fipronil used in vineyards to control ants can have acute toxic effects on Anagyrus sp. nr pseudococci and Coccidoxenoides perminutus, two microhymenopteran parasitoids of mealybugs (Mgocheki and Addison 2009). Thus, exposure of the biological control agents at release sites to baiting (especially aerial baiting where a fine dust is produced and a fraction of the bait is retained in the canopy) will be avoided. Coordination between field release and monitoring of biological control agents with National Parks staff involved in chemical control of yellow crazy ant supercolonies will be critical during this phase of this project. Training on release methodology and criteria will be provided to National Park personnel. Some additions to the Islandwide Survey (e.g., determination of host tree species composition, inspection of understorey for Tachardina and soft scale insects) will be made to facilitate selection of release sites. Interrogation of the survey database to identify the baiting history at waypoints will be an essential precursor to release of the biological control agents. The National Parks field crew will receive training in identifying and collecting scale insects and parasitoids.

C. Establishment and evaluation The absence of effective, quantitative monitoring for the establishment, spread and impact of most introduced biological control agents has been the Achilles’ heel of many biological control programs (McEvoy 1996). Estimation of the success or failure of many past biological control programs has relied on subjective measures, often post hoc expert opinion alone (e.g., DeBach et al. 1971, Greathead 1989, Griffiths and Julien 1998). For biological control on Christmas Island, protocols to quantify the establishment, population status, spread, and impact of biological control agents are essential.

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Two approaches will be used. First, a field experiment will be conducted using a Before-After- Control-Impact design to determine the establishment and population dynamics of the agents, and the effect of their release on host scale densities (counts per length of stem or per leaf) and parasitization rates, both in the canopy (random sampling of host plant material collected using a shotgun) and in the understorey (from saplings of known host trees), and abundance of yellow crazy ants (using counts on tree trunks and on the forest floor) at release and control sites before and after release of biological control agents. Sites (each 2-4 hectares) would be sampled 4 times before release of the agents and 4 times afterwards at two monthly intervals. Results will be analyzed as a one-way repeated measures ANOVA, using release of the biological control agents as the main factor, and comparing response variables before and after release. In this design, the time x treatment interaction is the key term, with a significant difference in response variables after, but not before release. Thus, this experiment at the forest plot scale would establish both the outcome of the release and the mechanism(s) driving any change in yellow crazy ant abundance. Second, at the much broader, island-wide scale the outcome of agents releases on yellow crazy ant supercolonies will be determined by comparing changes trunk traffic and ground activity (using card counts) of yellow crazy ants at four-month intervals at replicated release and control sites across the island. The number of control sites will be determined based on the release sites and area availability. Ideally, control sites should be distant enough from release sites so that the chances for biological control agent dispersal are low for a reasonable period of time. It will be necessary to determine how many of the selected release sites will be actually available for releases and the available areas for control sites where no other management practices for yellow crazy ants (i.e., application of toxic ant baits) will be applied. Spread of the biological control agents beyond release sites will be determined by placing potted ‘sentinel’ host plants, infested with Tachardina or coccoid scales, at set distances (probably at a logarithmic scale) from replicated release points, followed by their later collection to determine parasitization rates with distance from each release point. It may also be feasible to use the biennial Islandwide Survey to document spread of the biological control agent, at least onto understorey seedlings and saplings, at waypoints surrounding release sites. D. Monitoring for any non-target effects. As part of the monitoring program for of the establishment, population build-up, spread, and efficacy of Tachardiaephagus somervillei in parasitizing its target host, the yellow lac scale, any parasitism by T. somervillei on co-occurring kerriid scale Paratachardina pseudolobata (Kerriidae) and soft scales (Coccus and Saisettia spp.) will be examined. At each sampling date and site for T. somervillei, replicated branch samples with P. pseudolobata and leaf samples with soft scales will be collected and held in containers in the laboratory. Identity of any parasitoids that emerge will be determined.

All methodologies, from mass rearing to site selection for field release, monitoring, and evaluation will be adapted as experience and new knowledge is acquired. For example, the most rapidly evolving aspect of the release and monitoring program is likely to be the number and location of release points, based on the capacity of the production facility to provide parasitoids and their establishment and dispersal dynamics during the initial releases.

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Adapting methodologies will involve scientists and technical staff as well as the natural resource manager and park manager. CASAP, the scientific panel that advises Parks Australia on the management of the yellow crazy ant on Christmas Island, meets biannually to evaluate the management program and make recommendations to adapt the program as needed. There is flexibility for more frequent meetings as the need arises (Green and O’Dowd 2009).

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SECTION 7 Assessment of the likely consequences of not proceeding with the action to the environment, including people and communities, flora and fauna species, the rainforest ecosystem and Ramsar wetlands.

[Some of these issues were addressed in the original Environmental Referral (ER Part 2.2.1 Alternatives; RA Part A Risk Ref/ID 1, pp. 21-22]

An ambitious, forwarding looking biodiversity conservation plan for Christmas Island is framed around a long-term vision of retaining "Resilient ecosystems with self-sustaining populations of native species" (Director of National Parks 2014b). It recognizes the uniqueness of the natural environment of Christmas Island in a global arena and the need to protect its biodiversity. The scope of the plan covers: (a) native terrestrial species listed (or considered for listing) as threatened under the EPBC Act; (b) species which have an important role in the ecological integrity of the island's ecosystem; (c) species of conservation concern; (d) all endemic vertebrate species; (e) species which have a high level of international and community conservation interest; and (f) ecosystems and habitats critical for the survival of significant species, including the maintenance of critical ecological processes. The Plan has four key objectives: (1) maintain the ecological integrity of forest ecosystems; (2) maintain or increase populations of significant species; (3) maintain the ecological character of Ramsar wetlands; and, (4) contribute to maintaining groundwater ecosystems. The Plan further identifies four Key Threatening Processes (three of which are listed KTP) and a suite of Major Threat Risks that apply to the island. Of these, yellow crazy ants and their associated scale insects comprise both a Key Threatening Process and a Major Threat Risk whose effective management is identified as essential to the recovery of many species on Christmas Island. In fact, the documented impacts of supercolonies of the yellow crazy ant are so broad that they encompass every aspect of the scope of the Plan (a-f above) and three of its four key management goals (Objectives 1-3) above. The suppression of high- density supercolonies of the yellow crazy ant is central to three of these four objectives to conserve and restore biodiversity on Christmas Island. The most likely consequence of not proceeding with the action in the Environmental Referral is the failure to achieve fully Objectives 1-3 in the Draft Christmas Island Biodiversity Conservation Plan (2014). The recurrent costs of chemical control will continue to divert financial and human resources from other key biodiversity conservation objectives (see Beeton et al. 2010). Not proceeding with the actions proposed in the Environmental Referral will ensure that any management of supercolonies of the yellow crazy ant and amelioration of its impacts continues to rely completely on surveillance, monitoring, and chemical control but only as long as recurrent funding is available. The Expert Working Group (Beeton et al. 2010), recommended (Recommendation 8, High priority) that baiting with Fipronil continue but only as a short-term control measure. This recommendation was supported in the Australian Government Response. The use of Fipronil baiting is perpetually reactive because it relies on intensive, extensive and expensive field survey, primarily through the biennial Islandwide

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Survey (Green and O'Dowd 2009) to keep abreast of supercolony formation. This is especially difficult on a remote, oceanic island. Furthermore, chemical baiting has not been able to deal with incipient supercolonies where yellow crazy ant densities are increasing but still not high enough for the yellow crazy ant to monopolise the bait, and thereby minimise non-target impacts. Despite no evidence for the bioaccumulation of Fipronil or its metabolites in baited areas (CESAR Consultants 2011, 2013), there is still disquiet among all the stakeholders over its continued use, but a grudging acceptance of the lack of alternatives (Beeton et al. 2010). For all of these reasons, business-as-usual baiting with fipronil is seen as unsustainable in the long term and a transitional measure until biological control or some other alternative can be developed (Beeton et al. 2010, Recommendation 9 [High priority]; also see EPBC Referral, Helicopter baiting of the exotic yellow crazy Anoplolepis gracilipes supercolonies on Christmas Island, Indian Ocean, 2012). If the chemical baiting program is transitional and short-term (Beeton et al. 2010 and Australian Government Response), then the most likely longterm consequence of not proceeding with the actions detailed in this Environmental Referral is doing nothing. Comments on relevant controlling provisions for the proposed action: Wetlands of international importance. The principal threat to the two Ramsar wetland of international significance on Christmas Island, Hosnies Spring (202 ha) and The Dales (580 ha), is the impact of high-density supercolonies of the yellow crazy ant and their yellow lac scale mutualists (see the annotated Ramsar List 2013 - http://www.ramsar.org/cda/en/ramsar-documents-list-anno-australia/main/ramsar/1-31- 218%5E16713_4000_0__) (see also Section 2A). Not proceeding with the actions identified in this Environmental Referral would most likely mean continued reliance on chemical control measures to control supercolonies of the yellow crazy ant as outlined in Section 2A above. Moreover, Fipronil, because of its toxicity in aquatic ecosystems, cannot be used in the core areas of the wetlands to suppress these supercolonies (52 ha at Hosnies Spring and 137 ha at The Dales, respectively). Over the longterm, continued degradation of these wetlands could compromise the status of Hosnies Spring and The Dales as Ramsar wetlands.

Listed threatened species and communities, listed migratory species, and flora and fauna species. The yellow crazy ant Anoplolepis gracilipes is listed as a Key Threatening Process on Christmas Island under the EPBC Act 1999 (http://www.environment.gov.au/node/14577). Furthermore, the Expert Working Group (Beeton et al. 2010) concluded that the association of the yellow crazy ant with scale insects is the key factor generating the threat. Twelve EPBC Act listed species are given as threatened by yellow crazy ants on Christmas Island (Table 2.1). A broader perspective on threats to Significant species of which listed threatened species is a subset, is given in the draft Christmas Island Biodiversity Conservation Plan [Director of National Parks 2014b. The criteria for inclusion as a Significant Species as a EPBC Act listed species or flora and fauna species is given above and in Table 2.2 In the short-term, the most likely consequence of not proceeding with the actions detailed in this Environmental Referral would mean continued dependence on the use of a broad

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spectrum insecticide, Fipronil, with known non-target impacts, to suppress supercolonies of the yellow crazy ant and mitigate their known and suspected negative impacts on EPBC Act listed species, listed migratory species, and flora and fauna species. Because supercolonies can reform after chemical baiting and emerge elsewhere in the forest, this is a process that will have high recurrent costs (e.g. financial and human resources, non- target effects). In the longer term, the most likely consequence of not proceeding with this action is doing nothing.

People and communities. The most likely short-term consequence of not proceeding with the actions detailed in this Environmental Referral would be the continued of a broad- spectrum insecticide, Fipronil, to control supercolonies of the yellow crazy ant across many square kilometres of Christmas Island forest. In just about every community where broadscale aerial dispersal of insecticides has been used to control insect pests, its use has been controversial. This is so and likely to remain so, especially around Settlement, on Christmas Island. If chemical control is unsustainable and supercolonies of the yellow crazy ant persist, then the aesthetic, amenity, and cultural values of biodiversity in the island forest will continue to be degraded. Moreover, opportunities for broadening the economic base of the island economy, through development of nature-based tourism and recreation will be missed.

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Section 8 Details of comparable relevant introduction programs in Australia, overseas or on other islands, successful or otherwise, if available.

[Some of these issues were addressed in original submission (ER Part 2.2.1 Approval, p. 12)]

A. Scale insects as targets of biological control. Scale insects (Hemiptera: Coccoidea) have been the target of many, perhaps the majority, of biological control projects targeting insect pests (DeBach et al. 1971, Greathead 1989). This includes some of the most spectacular successes in the annals of biological control (e.g. control of cottony cushion scale Icerya purchasi - Caltagirone and Doutt 1989).

One of the clearest patterns in the historical record of biological control is that the greatest rates of success have been achieved against the Coccoidea and related sternorrhynchous Hemiptera (Mills 2006). Historically, the Coccoidea have dominated the scene as targets for biological control, accounting for nearly half of all projects in which some degree of success has been obtained (67% of all complete successes, 31% of all substantial successes, and 43% of all partial successes, as defined by post hoc expert opinion - see Section 6C above). Together with related the sternorrhynchous Hemiptera (e.g., and ) they account for about 2/3 of all successes in biological control of insects and for more than 4/5 of all projects in which complete success has been achieved (Clausen 1978).

The reasons why scale insects have been so frequently targeted in biological control programs are many. First, they comprise a disproportionately large proportion of introduced major pests (Greathead 1989). Small, sedentary and cryptic, they have been readily co-introduced into new areas with their many host plant species in the absence of effective quarantine barriers. Many are parthenogenetic so populations can establish from very few individuals. In the absence of many or most of their natural enemies in their native region, population densities in introduced areas can build up to threaten economically important crops or species of special conservation value. As such, there has been both an economic and conservation imperative to focus biological control efforts on the Coccoidea.

Life-history and ecological attributes of scale insects may be conducive to their successful biological control (Mills 2006). They are typically colonial, aggregative, and sedentary, with many generations per year on perennial, woody plant hosts, such that all life stages can be simultaneously present. Small size means that population densities per plant can be high. These attributes may facilitate population stability, giving parasitoids with many generations per year, like the encyrtid Tachardiaephagus somervillei, a broad window of host attack.

B. Biological control of scale insects on islands and in natural areas. Many programs for the biological control of scale insects have been conducted on islands, mostly in an agricultural or horticultural context. Over half of all biological control attempts in the BIOCAT database (Greathead 1989) have been on islands (1285/2484 records) and many of these have been defined as successful (DeBach 1962, Greathead 1989).

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Increasingly, introduced insect pests in natural areas, including national parks, have become targets of biological control (van Driesche et al. 2010, Van Driesche 2012). Most simply this is because few other control methods are applicable to broadscale control of insect pests that threaten the conservation value of natural areas, especially in remote locations like oceanic islands. When successful, biological control in natural areas, relative to other methods of control, is lower in cost, self-sustaining, and self-dispersing. An exemplar of successful biological control of a scale insect to prevent the extinction of a rare endemic plant in natural areas on a remote oceanic island is Fowler (2004).

C. Biological control of other Kerriidae. Other than the research described here, only one other attempt has been made to develop biological control for any other lac scale insect (Kerriidae). Research and development for the biological control of the lobate lac scale Paratachardina pseudolobata, native to Peninsular Malaysia and invasive on Christmas Island, in southern Florida, the Bahamas, Cuba, Puerto Rico, and recently Hawaii, commenced in 2003 but funding by the State of Florida USA was discontinued in 2008 before a suitable candidate for biological control was located and no releases have yet been made (Pemberton 2003, R.W. Pemberton, pers. comm. 2013). The lac insect of commerce Kerria lacca was deliberately introduced to Taiwan from Thailand in 1940 where it became a pest of arboriculture. Natural enemy exploration was conducted in Taiwan, but no further research was carried out (Chiu et al. 1985).

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73 1

1 Host specificity testing of Tachardiaephagus somervillei (Hymenoptera: 2 Encyrtidae), a biological control agent for the yellow lac scale 3 Tachardina aurantiaca (Hemiptera: Kerriidae)

4 G. Neumann1, D.J. O'Dowd2 and P.T. Green1 5 1Department of Botany, La Trobe University, Bundoora, Victoria 3086 Australia and 2School of 6 Biological Sciences, Monash University, Melbourne, Victoria 3800 Australia

7

8 Preamble

9 The yellow lac scale, Tachardina aurantiaca (Hemiptera: Kerriidae) is a damaging, invasive 10 pest on Christmas Island (Indian Ocean) and implicated in the formation of widespread, high- 11 density supercolonies of the invasive yellow crazy ant (Anoplolepis gracilipes) (O'Dowd et al. 12 2003, Abbott and Green 2007). Suppression and control of crazy ant supercolonies may be 13 afforded by the importation and release of Tachardiaephagus somervillei (Hymenoptera: 14 Encyrtidae), a widespread and abundant parasitoid of Tachardina that is known to parasitize 15 lac scales in its native range in Southeast Asia (Hayat et al. 2010, Green et al. 2013). Below we 16 provide a ‘desktop’ evaluation (Sands and Van Driesche 2004) of risk of importation and 17 release of T. somervillei on Christmas Island and then describe a protocol for host-specificity 18 testing recommending that it be conducted in the area of origin of Tachardina aurantiaca. 19

20 1. Evaluation of risk based on host records of Tachardiaephagus from the 21 scientific literature, hemipteran diversity on Christmas Island, and host 22 records of encyrtid parasitoids

23 To focus host-specificity testing for Tachardiaephagus somervillei, we used four approaches to 24 assess the risk of importation and release of the exotic biological control agent T. somervillei 25 to Christmas Island. All four lines of evidence indicate that there is a very low likelihood that 26 importation and release of T. somervillei would harm species of concern on Christmas Island.

27

28 1.1 Known host range of Tachardiaephagus somervillei

29 We evaluated known host species records for all species of Tachardiaephagus using the 30 Universal Chalcidoid Database (Noyes 2012) so that host range could be estimated and risk of 31 release of T. somervillei assessed for species of concern on Christmas Island. The Universal 32 Chalcidoidea Databases (Noyes 2012) is the most comprehensive database for chalcidoid 33 parasitoids, with over 120,000 host/associate records (including associations with food plants 34 of the hosts) and > 140,000 distribution records of the parasitoids in the superfamily 35 Chalcidoidea. It is very well developed, regularly updated and extremely well referenced. 36 Nevertheless, large databases can contain errors affecting reliability, such as erroneous 2

37 published host records and outdated parasitoid taxonomy (Kuhlmann et al. 2006). The most 38 important source of error in this database is actual published erroneous records. Since all 39 records are referenced, doubtful records can be investigated and if necessary, filtered out.

40 Seven described species of Tachardiaephagus are distributed in Southeast and South Asia, 41 and sub-Saharan Africa (Noyes 2012). Host records of Tachardiaephagus are restricted to 42 species within three genera (Kerria, Tachardina, and Paratachardina) of lac insects (Kerriidae) 43 (Table 1). Furthermore, all host species records for Tachardiaephagus somervillei are within 44 two genera: Kerria, comprising the lac insects of commerce, and Tachardina. These records 45 suggest that Tachardiaephagus somervillei is highly likely to have a narrow host range 46 restricted to kerriid host species.

47

48 1.2 Status of other scale insects on Christmas Island

49 We determined insect species on Christmas Island that could conceivably constitute non- 50 target species. Records for occurrence of scale insect species (Superfamily Coccoidea, the 51 same superfamily to which Tachardina aurantiaca belongs) on the island were compiled from 52 the literature and then supplemented by conducting >400 hours of structured search over 53 two years for endemic scale insects (Green et al. 2013). For specimens from the structured 54 search, all identifications were verified by Prof Penny Gullan (Australian National University), 55 a scale insect systematist.

56 A total of 24 species of scale insects in six families have been recorded on Christmas Island 57 (Table 2). All scale insects that could be considered as the most likely potential non-target 58 organisms are non-natives on Christmas Island and none are beneficial (Table 2). 59

60 1.3 Phylogeny of endemic hemipterans

61 The closest endemic relatives of the yellow lac scale on Christmas Island should be considered 62 to evaluate the risk of becoming non-target hosts of Tachardiaephagus somervillei. We 63 obtained a list of known endemic insect species on Christmas Island (James and Milly 2006) 64 and narrowed consideration of taxa on this list to the closest endemic relatives of lac scales 65 on Christmas Island in the Order Hemiptera, the same order to which the known target 66 species Tachardina aurantiaca belongs. These species comprise one true bug, a cicada, a 67 leafhopper, a spittlebug, and three planthoppers (Table 3). All of these endemic species occur 68 in different suborders (either suborder Auchenorrhyncha or Heteroptera) than the yellow lac 69 scale (suborder Sternorrhyncha).

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71

72 3

73 Thus, any potential for non-target impacts by T. somervillei on these endemic Hemiptera 74 would require a host range that bridges this very substantial phylogenetic distance (see above 75 and Fig. 2 in Cryan and Urban 2012), as well as distinctive morphologies, life-histories and 76 ecological attributes to its known host taxa in the Kerriidae. 77

78 1.4 Host ranges of encyrtid parasitoids known to attack members of the hemipteran 79 families with endemic species on Christmas Island.

80 The Encyrtidae (Hymenoptera, Superfamily: Chalcidoidea), the family to which T. somervillei 81 belongs, is one of the most important parasitic wasp (parasitoid) families for the biological 82 control of harmful insects, including a variety of scale insects infesting woody plants (Noyes 83 and Hayat 1994, Noyes 2012). The Encyrtidae currently comprises 460 genera and 3735 84 species in 2 subfamilies. The subfamily Encyrtinae includes 353 genera and 2920 species, 85 while the Tetracneminae includes 107 genera and 815 species. Approximately half of all 86 encyrtid species are associated with scale insects (Hemiptera: Coccoidea)(Noyes 2012). 87 Encyrtids are generally endoparasitoids meaning that the parasitoid egg is laid directly inside 88 the host’s body where the hatching larva completes development feeding on the host’s 89 tissue, ultimately killing the host. Encyrtids mostly parasitize immature life stages (or, rarely, 90 adults), but some species in one genus (Microterys) are egg predators (Noyes 2012).

91 We used the Universal Chalcidoidea Database to analyze all records of encyrtid parasitoid 92 associates of the hemipteran families represented by endemic species on Christmas Island. 93 Next, all recorded primary hosts of encyrtid species associated with these taxa were 94 determined. Consequently, any encyrtid species that share (as hosts) both coccoid species 95 (scale insects) and any family represented by endemic species on Christmas Island could be 96 found.

97 There are no known records in the Encyrtidae of any parasitoid species that attacks both scale 98 insects (Coccoidea) and host species in hemipteran families that have endemic 99 representatives on Christmas Island (Table 3). All known encyrtid primary parasitoids known 100 to attack species in families which have endemic representatives on Christmas Island have 101 primary hosts only within suborder Auchenorrhyncha. While species in these hemipteran 102 families are attacked by many species of chalcidoid parasitoids, many fewer or no encyrtid 103 species are known to use them as hosts, and not one encyrtid species is reported with a host 104 range so extremely broad that it encompasses both scale insects and any of these hemipteran 105 families. 106

107 2. Host specificity testing protocol

108 Actual records for host range of Tachardiaephagus and known patterns of parasitism in the 109 Encyrtidae indicate that it is highly improbable that T. somervillei would exhibit an extremely 110 broad host range (one that would be unprecedented in any single species of Encyrtidae 4

111 known to attack scale insects) and utilize any of the endemic hemipteran species on 112 Christmas Island.

113 2.1 Terminology

114 A ‘test species’ is an insect species that is tested as a potential non-target insect. Since no 115 non-target scale insects were identified on Christmas Island, the list of test species will be 116 determined using the centrifugal approach (Kuhlmann et al. 2006, Neumann et al. 2010). In 117 this approach, species other than the known target host of the biological control agent are 118 tested with the most closely related species (least phylogenetic distance, and with close 119 similarities in biology and ecology) tested first then less similar species thereafter (“centrifugal 120 principle” - Wapshere 1974).

121 A test species will be considered a ‘suitable host’ if parasitoid emergence is observed during 122 any of the exposure tests. To be a suitable host, the parasitoid must (a) accept the test 123 species when exposed, (b) must oviposit, (c) the eggs must hatch into larvae, (d) the larvae 124 must complete development using the test species as the resource, (e) the larvae must 125 pupate and, finally, (f) the emerging adult parasitoid must be able to exit (emerge from) the 126 test species. If parasitoid emergence from a test species is observed, the test species will be 127 considered a suitable host without assessing the viability, fecundity, sex ratio and other 128 characteristics of the emerging parasitoid generation.

129 A test species will be considered ‘susceptible’ if the parasitoid causes significant mortality due 130 to probing, host feeding (when the parasitoid stabs the insect with its ovipositor and then 131 feeds on the insect’s hemolymph), oviposition, or oviposition and larval development. Note 132 that even if some mortality is observed due to the above, the test species will only be 133 considered susceptible if the mortality resulting to the exposure to the parasitoid is 134 significantly higher than in control groups (negative controls) of the test species where the 135 test individuals are not exposed to the parasitoid.

136 The ‘parasitoid’ in this case is T. somervillei (Hymenoptera: Encyrtidae), a widespread 137 parasitoid native to Southeast Asia. It is known to parasitize some scale insects in the family 138 Kerriidae (lac scales) including the ‘target host’, Tachardina aurantiaca (O’Dowd et al. 2012).

139 2.2 Selecting test species

140 Host specificity testing can be a long and difficult process, depending on how problematic it is 141 to establish and manage colonies of the parasitoid, the target host, the host plant of the 142 target host, and the test species. Furthermore, resources and time are not unlimited and 143 permitting issues to collect insects and run field experiments (when needed) in Malaysia can 144 add to the time required to complete host testing. Therefore, a manageable number of 145 species should be used (10 to 15, but fewer are acceptable if high specificity is found initially). 146 The species used will be largely determined by availability, so determining the actual test 147 species at species or genus level is not possible at this point. We can, however, predict to 5

148 some degree the families of the test species that will be selected. We will focus on neococcid 149 taxa including the Kerriidae, the family to which the target host belongs (see Fig. 1 and Gullan 150 and Cook 2007). Considering the phylogenetic relationships of scale insect families (Fig. 1), 151 we will aim to test one species in the Kerriidae, Paratachardina pseudolobata, which is also 152 invasive on Christmas Island (Table 2), and more than one species from the Coccidae. Species 153 in the Diaspididae will also be considered as a less closely related group of scale insects. Early 154 in the host testing process, an ‘out-group’ (test species phylogenetically more distant) will 155 also be used, most likely selected from the Pseudococcidae (Fig. 1).

156 As mentioned above, availability will determine species selection to some degree but in some 157 of the groups (e.g. Coccidae) there may be a number of species available. Species that can be 158 reared in laboratory conditions would be preferred but carrying out tests in field conditions 159 should also be acceptable as long as the tests can be well controlled.

160 2.3 No-choice tests

161 In no-choice tests, only the non-target test species will be provided to the parasitoid in the 162 experimental replicates. This is the most stringent test method that places the parasitoid 163 under high “oviposition pressure”.

164 For each test species, trials will be replicated 10 times. Each replicate will consist of 1) one 165 experimental cage with the test species (50 individuals in three different age groups/instars; 166 150 individuals in total) exposed to 10 female and 10 male parasitoids, 2) one positive control 167 cage consisting of 50 mature female T. aurantiaca exposed to 5 female and 5 male parasitoids 168 in order to confirm that the parasitoids used in the experiment are of good quality and 169 capable of parasitism in the given exposure time, and 3) one negative control cage which will 170 be similar to the experimental cage except the test species will not be exposed to parasitoids. 171 The negative controls will be used to determine any effect of parasitoid exposure on insect 172 mortality other than successful parasitization such as probing, host feeding and oviposition 173 without successful parasitoid development.

174 The cages will consist of fine mesh bags (sleeves) fixed on the branches of plants that serve as 175 host plants for the scale insects. Parasitoids will be collected from a lab colony and 176 introduced to the cages < 24 h after emergence. Exposure time to parasitoids will be 177 determined during preliminary studies to determine the optimal time frame for mating, egg 178 maturation and oviposition by the parasitoid.

179 Evaluation:

180 1) If the positive control does not yield any parasitoid emergence, the replicate will not be 181 evaluated (failed replicate). 182 2) If the positive control yields parasitoid emergence the replicate will be evaluated. The 183 parasitization rate in the positive will be noted but will not be compared to parasitization 184 rates in the test replicate. 6

185 3) Parasitoid emergence in the test replicate will be noted along with the approximate age 186 and developmental stage of the individuals yielding parasitoid emergence. These 187 individuals will not be used to compare mortality rates due to reasons other than 188 successful parasitism. 189 4) Mortality of test species in test replicates and negative controls will be noted.

190 Analysis:

191 1) If any test replicates yielded any number of parasitoid emergences, the test species will 192 be considered a suitable host. The level of suitability of the test species will not be 193 compared to the suitability of the target host at this stage of the project. 194 2) Mortality (except mortality due to successful parasitism) rates of test insects and insects 195 in the negative control will be compared using 2-sample t-tests. If the mortality rate is 196 significantly higher (α = 0.05), the test species will be considered susceptible and the 197 reasons for mortality will be investigated.

198 If a test species is susceptible (but not suitable), behavioural observations will be made to 199 determine the cause of the effect of parasitoid exposure. The observational arena will be a 200 ‘window box’ arena. This arena is constructed by constructing a 10-cm W x 10-cm L x 5-cm H 201 box with one side being glass. The box is constructed so that it can be placed on the stem of a 202 potted host plant on which the test insects feed. Female parasitoids are then liberated inside 203 the box and the box closed. The potted plant then can be placed flat on its side and the box 204 positioned under a dissecting microscope. Video footage is then captured using a high 205 definition video camera with a microscope adaptor. The footage (4-6 h per observation) can 206 be analyzed later for the cause(s) of mortality (i.e. probing, host feeding, or oviposition 207 without full parasitoid development). If oviposition is suspected, dissections will also be 208 made to investigate parasitoid larval development.

209 2.4 Other testing methods

210 We considered using choice tests (where the parasitoid is presented with the test species and 211 the target host simultaneously) and sequential no-choice tests (where the parasitoid is 212 presented with the target host first then it is transferred on to the test species). However, in 213 our case these tests may have limited value for reasons given below.

214 A choice test would show whether a test species that is a suitable host in no-choice tests 215 would be consistently attacked when the target host is also available. A physiologically 216 suitable host may not be preferred over the target host and the assumption could be made 217 that parasitization would not occur (or at very low level) in field conditions. This test would 218 be used in cases where the suitable host (found suitable in a no-choice test) is of concern, e.g. 219 a beneficial or endemic non-target species that coexists with the target host. However, this 220 situation does not arise on Christmas Island where all known scale insects are non-native and 221 none of them are beneficial. This test is further limited by the assumption that the test 7

222 species and the target host occupy the same space hence providing the parasitoid with a 223 choice. This would also not be the case on Christmas Island.

224 A sequential no-choice test would further show the unsuitability of a test species. The 225 parasitoid may be ‘motivated’ to attack the test species even if it did not attack it in a simple 226 no-choice test if the parasitoid is first allowed to initiate oviposition behavior and to gain 227 experience. This test may be even more stringent than the simple no-choice test and one 228 would consider using it in the case of a test species that is of great concern to further show 229 the unsuitability of the test species. This would again require a beneficial or endemic non- 230 target species neither of which exists on Christmas Island. Furthermore, similar to the choice 231 test, this would assume either the coexistence or very close proximity of the test species and 232 the target host. 233

234 3. Options for the location for host-specificity testing

235 There are three options for the location where host specificity testing could be conducted: 1) 236 in quarantine containment at the release location (Christmas Island); 2) in quarantine 237 containment on mainland Australia; and 3) in the native geographical range of the biological 238 control agent where no containment would be necessary.

239 Option 1. Testing in containment at the release location (Christmas Island)

240 This option is the least attractive and involves increased risks and expenses. There is no 241 quarantine containment facility on Christmas Island, a remote oceanic island. The expense of 242 constructing such a facility to Quarantine Approved Premises Criteria (Quarantine Insectary 243 Level 2) for host-specificity testing of a single agent would be prohibitive. Even if there were 244 such a facility, in case of escape, the biological control agent would find an environment very 245 similar to its native range with its natural host T. aurantiaca in abundance. The benefits of 246 this option would include easy access to the natural host for the parasitoid colony and no 247 further travel if and when a release permit is obtained.

248 Option 2. Testing in containment on mainland Australia

249 While this option is more attractive than Option 1 above, it is still somewhat risky, and labour 250 and cost intensive. It brings few benefits. The biological control agent (parasitoid) would have 251 to be brought into containment along with its natural host (Tachardina aurantiaca). This 252 would pose the risk of not only a parasitoid escaping but also a potentially invasive, host- 253 generalist scale insect (on Christmas Island, for example, at least 15 horticultural species are 254 attacked by T. aurantiaca, including three species of Citrus, Macadamia, Guava, Pomegranate, 255 Chili, Eggplant, Star fruit, and Soursop)(R.W. Pemberton and D.J. O’Dowd, unpublished 256 results). Using a containment facility in the temperate region of Australia could decrease 257 these risks. In case of quality control issues of the biological control agent due to ‘lab 258 selection,’ additional travel, collection, and importation of the biological control agent would 8

259 be necessary, further increasing costs. Natural host colony loss (which can easily happen due 260 to overexposure to the parasitoid agent, fungal infections, etc.) would necessitate multiple 261 importations of the natural host, which would once again significantly increase costs and risk.

262 Option 3. Testing in the native geographical range of the biological control agent with no 263 containment necessary

264 In our case, this option is the most attractive, most cost-effective, and least risky. There are 265 many benefits to study biological control agents and carry out host specificity studies in the 266 native geographical range of the agent. In our case, both the parasitoid and its natural host is 267 readily available from the wild in Malaysia which ensures good quality parasitoid and host 268 cultures. The risk factor would be zero as there would be no importation of any organisms. 269 Although obviously desirable, such studies in the native range are not common, in part 270 because of the lack of local research facilities, lack of skilled and reliable cooperators, or 271 sufficient time in what are sometimes difficult locations (Van Driesche et al. 2008). However, 272 scientific work is cost-effective in Malaysia compared to Options 1 and 2 and our network of 273 collaborators is well established (see Table B1, O’Dowd et al. 2012). Options 1 (test in 274 containment on Christmas Island) and 2 (test in containment on the Australian mainland) 275 offer no benefits over this option. We therefore suggest that host specificity testing is carried 276 out in the native range of the biological control agent. This option carries the least risk and is 277 consistent with Recommendation 2 in a review of biosecurity risks in biological control 278 commissioned by the Australian Government (Ferrar et al. 2004) that biological control 279 practitioners undertake host specificity testing in the native region before any importation.

280

281 References

282 Abbott, K.L. 2004. Alien ant invasion on Christmas Island, Indian Ocean: the role of ant-scale 283 associations in the dynamics of supercolonies of the yellow crazy ant, Anoplolepis gracilipes. 284 PhD Thesis, Monash University, Melbourne, Australia.

285 Abbott, K. and P.T. Green. 2007. Collapse of an ant-scale mutualism in a rainforest on 286 Christmas Island. Oikos 116: 1238-1246.

287 Bellis, G.A., J.F. Donaldson, M. Carver, D.L. Hancock, and M.J. Fletcher. 2004. Records of insect 288 pests on Christmas Island and the Cocos (Keeling) Islands, Indian Ocean. Australian 289 Entomologist 31: 93-102.

290 Ben-Dov, Y. 2012. Scalenet (http://www.sel.barc.usda. gov/scalenet/scalenet.htm).

291 Campbell, T.G. 1968. Entomological survey of Christmas Island (Indian Ocean) with special 292 reference to the insects of medical, veterinary, agricultural and forestry significance. 293 Unpublished Report, CSIRO Division of Entomology. 48 pp. 9

294 Cryan, R.C. and J.M. Urban. 2012. Higher-level phylogeny of the insect order Hemiptera: is 295 Auchenorrhyncha really paraphyletic? Systematic Entomology 37: 7-21.

296 CSIRO. 1990. CSIRO Entomological survey of Christmas Island. Phase 2. Unpublished Report. 297 Australian National Parks and Wildlife Service Consultancy Agreement. 67 pp.

298 Ferrar, P., I.W. Forno, and A.L. Yen. 2004. Report of the Review of the Management of 299 Biosecurity Risks Associated with the Importation and Release of Biological Control Agents. 300 Australian Government Department of Agriculture, Fisheries and Forestry, Canberra, 301 Australia. 27 pp.

302 Green, P.T., D.J. O’Dowd, G. Neumann, and S. Wittman. 2013. Research and development of 303 biological for scale insects: indirect control of the yellow crazy ant (Anoplolepis gracilipes) on 304 Christmas Island, 2009-2013. Final report to the Director of National Parks, Parks Australia, 305 Canberra, A.C.T. 66 pp.

306 Gullan, P. J. and L. G. Cook. 2007. Phylogeny and higher classification of the scale insects 307 (Hemiptera: Sternorrhyncha: Coccoidea). Zootaxa 1668: 413-425.

308 Göllner-Scheiding, U. 1980. Revision der afrikanischen Arten sowie Bemerkungen zu weiteren 309 Arten der Gattungen Leptocoris Hahn, 1833, und Boisea Kirkaldy, 1910. Deutsche 310 Entomologische Zeitschrift, N.F. 27: 103-148.

311 Hayat, M., S. Schroer, and R.W. Pemberton. 2010. On some Encyrtidae (Hymenoptera: 312 Chalcidoidea) on lac insects (Hemiptera: Kerriidae) from Indonesia, Malaysia and Thailand. 313 Oriental Insects 44: 23-33.

314 James, D. and N. Milly. 2006. A biodiversity inventory database for Christmas Island National 315 Park. A report for the Department of Finance & Administration and Department of 316 Environment & Heritage. Director of National Parks, Australian Government, Canberra. 49 317 pages.

318 Kuhlmann, U., U. Schafner, and P. G. Mason. 2006. Selection of non-target species for host 319 specificity testing. Pages 15-37 in Bigler, F., D. Babendreier and U. Kuhlmann (eds) 320 Environmental Impact of Invertebrates for Biological Control of Arthropods: Methods and Risk 321 Assessment. CAB International, Oxford.

322 Linnavuori, R. 1975. Homoptera: Cicadellidae, Supplement. Insects of Micronesia 6. No. 9. 323 pp. 611-632

324 Neumann, G., P. A. Follett, R. G. Hollingsworth, and J. de León. 2010. High host specificity in 325 Encarsia diaspidicola (Hymenoptera: Aphelinidae), a biological control candidate against the 326 white peach scale in Hawaii. Biological Control 54: 107-113.

327 Noyes, J.S. and M. Hayat. 1994. Oriental mealybug parasitoids of the Anagyrini (Hymenoptera: 328 Encyrtidae) viii+554pp. CAB International, Wallingford, UK. 10

329 Noyes, J.S. 2012. Universal Chalcidoidea Database. World Wide Web electronic publication. 330 http://www.nhm.ac.uk/chalcidoids

331 O’Dowd, D.J., P.T. Green, and P.S. Lake. 2003. Invasional ‘meltdown’ on an oceanic island. 332 Ecology Letters 6: 812-817.

333 Prinsloo, G.L. 1977. On the encyrtid parasitoids (Hymenoptera: Chalcidoidea) of lac insects 334 (Hemiptera: Lacciferidae) from southern Africa. Journal of the Entomological Society of South 335 Africa 40: 47-72.

336 Prinsloo, G.L. 1983. A parasitoid-host Index of Afrotropcial Encyrtidae (Hymenoptera: 337 Chalcidoidea). Entomological Memoir of the Department of Agriculture Republic of South 338 Africa no. 60. 35 pages.

339 Sands, D.P.A. and R.G. Van Driesche. 2004. Using the scientific literature to estimate the host 340 range of a biological control agent. Pages 16-23 in Van Driesche R.G. and R. Reardon (eds.). 341 Assessing host ranges for parasitoids and predators used for classical biological control: a 342 guide to pest practice. University States Department of Agriculture Forest Health Technology 343 Enterprise Team, Morgantown, West Virginia USA. FHTET-2004-3.

344 Van Driesche, R., T. Center, M. Hoddle, and N. Mills. 2008. Can efficacy of new biological 345 control agents be predicted before their release? Pp. 1-13 in Mason, P.G., D.R. Gillespie, and 346 C. Vincent (eds.). Proceedings of the Third International Symposium on Biological Control of 347 Arthropods. Christchurch, New Zealand, 8-13 February 2008. United States Department of 348 Agriculture, Forest Service, Morgantown, WV, FHTET-2008-06, December, 2008, 636 p.

349 Wapshere, A.J. 1974. A strategy for evaluating the safety of organisms for biological weed 350 control. Annals of Applied Biology 77: 201-211.

351 Woods, B. and E. Steiner. 2012. Christmas Island fruit fly and scale survey. Report to 352 Department of Agriculture and Food, Government of Western Australia. 22 pp. 353 11

354 Table 1. Records of known host families and genera for the primary parasitoid 355 Tachardiaephagus (Encyrtidae). Taxonomy follows Prinsloo (1977). The biological control 356 agent under investigation, Tachardiaephagus somervillei, is in bold. As a genus, 357 Tachardiaephagus has an extremely broad geographic range. With the exception of one 358 probably erroneous host record in Africa (Prinsloo 1983), all Tachardiaephagus species appear 359 to be family specialists and restricted to the Kerriidae. For host genera, number of species 360 recorded as hosts is in parentheses. Based on Noyes (2012, Universal Chalcidoidea Database, 361 http://www.nhm.ac.uk/research-curation/research/projects/chalcidoids/database/), except 362 for records for T. somervillei and T. sarawakensis (Hayat et al. 2010; Green et al. 2012; R.W. 363 Pemberton, pers. comm.)

Parasitoid species Distribution Recorded hosts (all Kerriidae)

Tachardiaephagus India, Malaysia, Thailand Kerria spp. (4)1 somervillei Tachardina aurantiaca Tachardina sp.2 T. sarawakensis Sarawak (East Malaysia) Tachardina aurantiaca

T. tachardiae Brunei, China, India, Indonesia, Kerria spp. (8) Malaysia, Sri Lanka, Taiwan, Paratachardina lobata Vietnam, Azerbaijan T. similis Afrotropical, South Africa Tachardina sp. (1)

T. absonus Afrotropical, South Africa Tachardina spp. (2)

T. communis Afrotropical, South Africa Tachardina spp. (5)

T. gracilis Afrotropical, South Africa Tachardina sp. (1)

364 1In Noyes (2012) both Kerria and Laccifer species are listed as hosts. However, Scalenet (Ben- 365 Dov et al. 2012) indicates that Laccifer is a synonym for Kerria, so we have synonymized these 366 records with Kerria species.

367 2 Probably T. aurantiaca, since it is the only known Tachardina species in Asia. 12

Table 2. Scale insects of Christmas Island. It is highly probable that all of these species, with broad host plant ranges and geographic distributions, are non-native to Christmas Island and introduced following human settlement. The target species, Tachardina aurantiaca, for biological control is in bold. Families are arranged in increasing phylogenetic distance from the Kerriidae based on Gullan and Cook (2007). All scale insect taxa are 'neococcids' except for Icerya purchasi ('archeococcid'). Taxonomy and distributions from Ben-Dov et al. (2012), http://www.sel.barc.usda. gov/scalenet/scalenet.htm).

Family and Species1 Common Name Distribution

Kerriidae (lac scales) Paratachardina pseudolobata False lobate lac scale Oriental, Nearctic, Neotropical (Kondo & Gullan) Tachardina aurantiaca (Cockerell) Yellow lac scale Oriental Coccidae (soft scales) Ceroplastes ceriferus (Fabricius) Indian wax scale Cosmopolitan C. destructor Newstead White wax scale Afrotropical, Australasia, Oriental Coccus celatus De Lotto Green coffee scale Afrotropical, Australasia, Oriental C. hesperidium Linnaeus Brown soft scale Cosmopolitan Milviscutulus mangiferae (Green) Mango shield scale Cosmopolitan Parasaissetia nigra (Nietner) Nigra scale Cosmopolitan Pulvinaria urbicola Cockerell Urbicola soft scale Pantropical P. psidii Maskell2 Green shield scale Cosmopolitan S. coffeae (Walker) Black scale Pantropical Saissetia oleae (Olivier) Hemispherical scale Cosmopolitan Diaspididae (armoured scales) Aspidiotus destructor (Signoret) Coconut scale Cosmopolitan Hemiberlesia palmae (Cockerell) Tropical palm scale Cosmopolitan (Signoret) 2 Black thread scale Cosmopolitan Lindingaspis sp. -- -- Pseudaulacaspis pentagona White peach scale Cosmopolitan (Targioni Tozzetti) Unaspis citri (Comstock) White louse scale Cosmopolitan

Cerococcidae (ornate pit scales) Cerococcus indicus (Maskell) Spiny brown coccid Cosmopolitan

Pseudococcidae (mealybugs) Dysmicoccus finitimus Williams Asian coconut mealybug Australasia, Oriental

Ferrisia virgata (Cockerell) Striped mealybug Cosmopolitan Nipaecoccus viridis (Newstead) Spherical mealy bug Cosmopolitan Pseudococcus longispinus Long-tailed mealy bug Cosmopolitan (Targioni Tozzetti)

Monophlebidae (giant scales) Icerya purchasi (Maskell) Cottony cushion scale Cosmopolitan

1Records from Campbell (1968), CSIRO (1999), O'Dowd et al. (2003), Bellis et al. (2004), Abbott (2004), Woods and Steiner (2012) and Neumann et al. (unpubl. results); 2Tentative identifications 13

Table 3. Endemic hemipteran species known from Christmas Island and primary parasitoids (superfamily Chalcidoidea: family Encyrtidae) associated with the families represented by the endemic species. The data were extracted from the Universal Chalcidoidea Database (Noyes 2012). The families Nogodonidae and Rhopalidae have no associated chalcidoid primary parasitoids and therefore endemic species in these families on Christmas Island can most likely be excluded from all further consideration. Cicadellidae and Delphacidae have the highest diversity of chalcidoid primary parasitoids but have magnitudes lower diversity of encyrtid primary parasitoids. These data suggest that the encyrtid primary parasitoids of families with endemic species on Christmas Island appear to not have host range overlap with taxa where the target lac scale is included and the host range separation is at the suborder level suggesting very distant phylogenetic separation. During the database analysis, only records with species-level chalcidoid identification were used. N/A indicates not applicable.

No. chalcidoid No. encyrtid 1o Suborder/Family host associates parasitoid range of encyrtids Endemic of family species of parasitizing family species Family family

Xestocephalus Cicadellidae 627 6 Auchenorrhyncha Izzardi1 (Cicadellidae) Oxypleura Cicadidae 35 0 N/A calypso Clovia eximia Cercopidae 71 4 Auchenorrhyncha (Cercopidae- Aphrophoridae) Ugyops aristella Delphacidae 248 5 Auchenorrhyncha (Delphacidae- Cicadellidae) Varcia Nogodinidae 0 N/A N/A flavicostalis Salona oceanica Leptocoris Rhopalidae 0 N/A N/A 2 subrufescens 1 Xestocephalus izzardi is also reported from Palau in the western Pacific Ocean (Linnavuori 1975). Its status as an endemic on Christmas Island is questionable. 2 Leptocoris subrufescens on Christmas Island has been classified to subspecies status (L. subrufescens subrufescens,). Another subspecies (L. s. flava) is described from Yap, western Pacific Ocean (Göllner- Scheiding 1980). More research is needed to resolve the taxonomic status of these two subspecies of L. subrufescens.

14

Pseudococcidae

Coccidae

Kerriidae Diaspididae

Gondwanan clade

BSE clade

Asterolecaniidae Acanthococcid group

Dactylopiidae

Figure 1. Portion of a phylogram of the scale insects based on nucleotide sequencing of nuclear 18S rRNA for 72 species of scale insects and 10 outgroup taxa (see Gullan and Cook [2007] for details). Only the neococcoids (red) are shown here. For all families given here (except the Kermesidae), Bayesian posterior probabilities (numerical values above branches) are >90%. For a host test list, we will select species from those families given in bold. The lac scales Kerriidae (bold and italics) is a sister family of the soft scales Coccidae, more distant to the armoured scales Diaspididae, and even further removed from the mealybugs Pseudococcidae.

Comments and suggestions for host specificity testing protocol (Neumann et al. 2013) from 5 ad hoc reviewers

Dr. Bob Pemberton (USDA-ARS, Invasive Plant Research Laboratory, Ft. Lauderdale, Florida) Leading proponent of safety in classical biological control of weeds and insects; development of biological control for insect pests and weeds, including the lobate lac scale, foreign exploration for natural enemies)

You all have done an excellent job on your testing proposal! I have attached the reviewed doc which has only a few comments. 1. More information on T. somervillei. 2. Say that it attacks only attached females not the smaller attached males. 3. Some reviewers will be unfamiliar with the basic biologies of scales and parasitoids so it would be helpful to describe the life history of your lac scale. Then what stages the parasitoid has been reared from, and if known, what stages are attacked. 4. If it is known what host plants that Tachardina aurantiaca was on when parasitized by T. somervillei and list some. 5. Say something about the phenologies of both the scale and parasitoid to indicate that they overlap or that both appear to have continuous populations because of the moist tropical conditions. This would suggest that there should by synchrony and climatic suitability on Christmas Island. 6. Indicate that there is no parasitism of the scale on Christmas Island but that it is common or usual in its native Malaysia. Give some parasitism rates found. More needs to be said about the potential of the parasitoid and probable establishment based on similarities of the native area and Christmas Island. This information helps justify the host range testing. 7. For the use of "suitable host" some people say "suitable laboratory host" or "potential host." Could use "suitable physiological host" (note: same comment made by Barbara Barratt). 8. Although you say containment would not be needed in Malaysia, a containment level that excludes hyperparasitoids is needed.

Dr. Barbara Barratt (Principal Scientist, AgResearch Invermay, Biocontrol, Biosecurity and Bioprocessing, Mosgiel, New Zealand) Leading researcher in host specificity testing of parasitoids, risk assessment for classical biological control, biocontrol in natural areas, and developed the BIREA for New Zealand

Basically looks pretty good to me but I have posed one or two questions. 1. Line 11. What damage is caused by T. aurantiaca? 2. Line 42. Clarify "actual published erroneous records." 3. Line 64. Revise subheading so as to not prejudge phylogenetic status 4. Line 71. "They would seem to be a real long-shot!" 5. Line 132. Add Wapshere 1974 as reference for centrifugal testing. 6. Line 140. Clarify - this determines physiological host range as distinct from ecological host range. 7. Line 154. Add "and the host plant of the target" to the issue of establishment and maintenance of colonies of the parasitoid. 8. Line 198. Rationale for not comparing parasitization rates in the control to the test replicate. 9. Line 206. Same issue as at Line 198. 10. Line 208. Clarify reasoning here. 11. Line 292. Are coccid and diaspidid non-target species you want going to be available in Malaysia?

Dr Matthew Purcell (Director, USDA-ARS Australian Biological Control Laboratory, Brisbane) Leading researcher in classical biological control of weeds.

I’ve gone through the document a few times and it seems bullet proof to me. Given the remote location of Christmas Island to Australia, and to anywhere else for that matter, the risks are very low. Given that you have looked thoroughly into the insect fauna of the Island and that no endemic scales exist, your host testing protocol is more than adequate and fully justified. 1. It may be appropriate to mention in the Preamble that host-specificity testing is recommended to be conducted in Malaysia. 2. In section 2, I think you should make it implicit that only scales are being tested. 3. In section 2.2, it may be best to explain why it is not possible to determine the species of scale insects to be tested at this point in time. 4. In section 2.3, 2nd paragraph, mature adult females are obviously the preferred stage for the parasitoid to attack. It may be worth discussing here or elsewhere a little about the biology of the host and its parasite, especially since most Encyrtidae attack immatures. This would be particularly useful information when evaluating the experimental procedure. 5. In section 2.3, Evaluation 1. It would be beneficial to explain why parasitization rate in the positive control will not be compared to parasitization in rates in the test replicate. Ditto for Evaluation 3.

Dr. Don Sands OAM (retired, CSIRO, Ecosystems Sciences, Brisbane, QLD) Leading researcher in the biological control of scale insects and biological control of weeds

I could add very little to your thorough testing protocol - you covered it very well. I have attached a few thoughts and comments that might be included or considered. 1. A quarantine facility on Christmas Island should be adequate for needed studies of specificity. Adequate lighting allowing sunlight to enter through double-glass will minimise artefacts in parasitoid behaviour. 2. Light and cage materials may be important when host testing and breeding parasitoids in cages. Encyrtidae often use light, certain spectra, intensity & absorption as ovipositional stimuli and for host recognition. I recommend black cages be used, as now used for rearing parasitoids by USDA and elsewhere, to avoid false positives and negatives. 3. Host specificity testing of parasitoids. Based on indigenous Hemiptera species and parasitoid host range only a small precautionary list of non-targets species would appear to require testing. As exotic scales on Christmas Island would appear to be the closest relatives to the target lac, these could be easily included in preliminary tests. 4. Many encyrtid parasitoids behave differently to their arthropod hosts feeding on different plant hosts. Tachardiaephagus could be tested with scales attached to a small range of plants, particularly native species. 5. Retain vouchers from each original shipment of wasps and also second generation of lab. – reared wasps, and have permanent preserved samples of insects released on the island. 6. Choice tests (target and non-target in same cage) can give misleading information and incorrect results from the diffusing pheromones derived from host affecting non-target host identity. 7. Nursery & mass rearing parasitoids for distribution. Once approvals for release minimise the numbers of parasitoids and number of their generations, in the confinement of quarantine, to avoid lab-adaptation or bottle-necking (can happen in 4 generations). 8. Mass rearing post approval is best done in cages outside of quarantine and with hosts + parasitoids exposed to diurnal temperature fluctuations, to enhance climate pre-adaptation and maximise numbers of parasitoids for release. 9. Release of parasitised scales on live plants (or bouquets) is more effective than the release of adult parasitoids. 10. There may be unrecognised hyper-parasitoids on Christmas Island likely to attack the Tachardiaephagus after it is introduced – worth predicting this possibility in case it influences parasitoid efficiency. 11. Release methods and localities, post release and establishment monitoring might best be outlined and methods included (& for what period) in the protocol?

Dr Tim Haye (Research Scientist, Centre for Agricultural Bioscience International [CABI], Rue des Grillons 1, CH-2800, Delemont, Switzerland) Leading researcher on the biology and use of parasitoids in classical biological control of insect pests

I think the approach you suggest is well done. For us here at CABI it is a common practise to test the parasitoids in their native range. I do this for all the European insects that got introduced to North America. As lab tests often overestimate the host range of parasitioids, you may consider to look at the ecological host range (field host range) of your biocontrol agent in Malaysia. I would just take some samples from sites where maybe your target and non-targets co-occur. This would back up your lab data in case you may get non-target parasitism. The chalcidoid data base is a good tool, but the host records are often not complete, so any additional field data would make a later petition for releases of the biocontrol agent much stronger.

The only thing I would hesitate about in your test design is that you are planning to test groups of parasitoids instead of individual wasps. Is there a specific reason for this? When testing groups you may have competition of parasitoids which may negatively influence your test outcome. Is there much known on the biology of the wasps (fecundity, pre-oviposition period, longevity)? All these factors may influence the test outcome, but I assume most of this is not known?

RESEARCH AND DEVELOPMENT OF BIOLOGICAL CONTROL FOR SCALE INSECTS: INDIRECT CONTROL OF THE YELLOW CRAZY ANT ON CHRISTMAS ISLAND, 2009‐2013

Peter T. Green, Dennis J. O’Dowd, Gabor Neumann, and Sarah Wittman Department of Botany, La Trobe University, Bundoora, Vic 3086

Final Report to the Director of National Parks

3 July 2013

1

Executive Summary

Research Project 1. YCA dependence on honeydew

Project 1a. Honeydew use by YCA. Successful biological control of the yellow lac scale Tachardina aurantiaca could remove a large fraction (an average of 70% to an average of 87%, depending on assumptions) of honeydew available to the yellow crazy ant. However, there is considerable site‐to‐ site variation in the likely contribution of T. aurantiaca to the total honeydew economy, and it is not certain that targeting this species alone would provide adequate indirect control for the YCA in all supercolonies. The most prudent course of action is to target the entire assemblage of honeydew‐ producing scale insects through the introduction of a biological control agent for T. aurantiaca, complemented by the use parasitoids already present on Christmas Island to target the coccoid soft scales. Project 1b. Stable Isotope Analysis. Stable isotope analyses of YCA workers, plants, herbivores and predators collected from four declining supercolonies in 2010‐2011 indicate that at supercolony densities, a substantial fraction of YCA dietary intake is plant‐derived. This is consistent with the idea that YCA supercolonies depend heavily on honeydew derived from scale insects for a large fraction of colony food and energy requirements, and provides support for the idea that indirect control over YCA supercolonies could be achieved by targeting honeydew‐producing scale insects for biological control. Project 1c. Scale Insect Removal Experiment. The exclusion of YCA from access to scale insects at a large experimental field site caused YCA activity on the ground to decline 5‐fold within 4 weeks, compared to pre‐treatment levels. This large field experiment validates the key concept of indirect biological control for YCA on Christmas Island; exclusion of honeydew‐producing scale insects from YCA caused a significant and substantial reduction in YCA abundance on the ground. Project 1d. Carbohydrate supply and YCA growth and behaviour. Dynamics and behaviour of YCA in laboratory colonies depended on carbohydrate supply. When sugar supply was elevated, reproductive output by queens increased, death rates of workers decreased, foraging tempo quickened, and interspecific aggression intensified. These results suggest that sugar supply, through honeydew supplied from scale insects, plays an important role in YCA supercolony dynamics, foraging efficiency, and interspecific aggression.

Research Project 2. Scale insects and natural enemies Projects 2a and 2c. Natural enemies survey, parasitization rates of lac scale insects (Tachardina aurantiaca and Paratachardina pseudolobata) and parasitoid behaviour in area of origin (Southeast Asia) and on Christmas Island. Few natural enemies of the key honeydew‐producing scale insect Tachardina aurantiaca occur on Christmas Island, and they do not regulate its population size. No parasitoids of female T. aurantiaca were found. Conversely, within its native range in Malaysia, T. aurantiaca is rare and patchily distributed, associated with diverse natural enemies, including at least five species of primary parasitoids, and it suffers high parasitization rates – all attributes consistent with population regulation by natural enemies. Importantly, high parasitism rates in Malaysia occurred in the presence of honeydew‐collecting ants, including the yellow crazy ant. Of the primary parasitoids known to attack yellow lac scale from our studies in Malaysia,

2

Tachardiaephagus somervillei (Encyrtidae) is the most promising agent for introduction and release on Christmas Island. All Tachardiaephagus species have a narrow host range and appear to be family specialists, known only to attack the Kerriidae, the family to which the yellow lac scale belongs (Table 3). Our initial studies indicate that (a) T. somervillei attacks T. aurantiaca across 1900‐km range in Peninsular Malaysia and Malaysian Borneo, (b) is the most abundant natural enemy of T. aurantiaca, (c) has a short life cycle compared to its host, (d) exhibits superparasitism (i.e., where multiple progeny emergence from a single host individual); (e) causes high rates of parasitism on T. aurantiaca in the presence of tending ants, including the yellow crazy ant, and (f) can be reared under laboratory conditions. Coccophagus ceroplastae and Encyrtus infelix, both parasitoids of a wide variety of coccid scale insects, are already present on Christmas Island. The three parasitoids T. somervillei. C. ceroplastae and E. infelix could be deployed in combination against the entire assemblage of honeydew‐producing scale insects in YCA supercolonies. Project 2b. Scale insect survey on Christmas Island (biology, host range, habitats, natural history, identification). No native or endemic scale insect species have been discovered in intensive and extensive searches for scale insect species on Christmas Island. However, 400 hours of search over two years did yield five additional exotic scale insect species previously unknown to the island. Assuming that any prospective biological control agent for Tachardina aurantiaca on Christmas Island would be a narrow family specialist (Kerriidae) and all known scale insect species are non‐ native and invasive, the probability of any direct non‐target effects is negligible. Project 2d. Genetic and morphological matching between Tachardina aurantiaca and Paratachardina pseudolobata on Christmas Island and Southeast Asia. The single population of T. aurantiaca on Christmas Island matches morphologically and genetically those populations examined so far within its native region in Southeast Asia. This establishes the identity of Tachardina aurantiaca on Christmas Island, making the successful establishment on Christmas Island of a biological control agent that attacks T. aurantiaca in its native range more likely. Further collections and analyses of T. aurantiaca in Eastern Malaysia (Sabah) and exploration in southern Thailand should establish if any morphological or genetic variation occurs across its native distribution in Southeast Asia. Project 2e. Scale population dynamics, especially the ecology of lac scale, Tachardina aurantiaca. The life cycle of Tachardina aurantiaca is relatively long for a scale insect but several overlapping generations occur in a single year. Female size is significantly correlated with fecundity, indicating that conditions that affect female size are likely to have a strong influence on population growth rates and density. The role of males in reproduction is not yet clear although it appears that some females can reproduce parthenogenetically. All of these attributes will influence host‐parasitoid dynamics on Christmas Island.

Research Project 3. Strategic planning for biocontrol introduction

Project 3a. Planning research in step with legislative and regulatory requirements for biological control introductions. Two regulatory frameworks govern the importation and release of an exotic biological control agent on Christmas Island. To gain approval to import and release a biological control agent on Christmas Island, we will need to prepare and submit two sets of documents: an Approvals Package under the DAFF protocol and an Environmental Referral to EACD, DSEWPaC. The key government contacts involved in the two frameworks are described and currently consulting

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each other. Project 3b. Identification and agreement with co‐operators. Collaborations have been established with researchers with expertise in the systematics, biology, and molecular genetics of scale insects, taxonomy of microhymenopteran parasitoids, and biological control of scale insects. Cooperative agreements have been established with the Forestry Research Institute of Malaysia (FRIM) and Sarawak Forestry that provide benchspace and technical assistance. Dr Neumann is serving as an advisor of a Masters student (Universiti Sains Malaysia, Penang) who is conducting research on this project at FRIM. Research and collecting permits have been obtained from the States of Sarawak and Sabah, as well as a national collecting permit. Project 3c. Safety and sanitary protocols anticipating a pre‐release environmental assessment of introduction. An agreed, peer‐reviewed host list and host specificity protocol has been prepared for evaluating the risk of introducing T. somervillei to Christmas Island to control the invasive yellow lac scale, Tachardina aurantiaca. It is recommended that host specificity testing be conducted in the area of origin of Tachardina aurantiaca. A protocol for ensuring that hyperparasitoids are not accidentally co‐introduced with importation of T. somervillei to Christmas Island has also been prepared. Project 3d. Rearing, release and monitoring protocols. The requirements to establish a founder population of Tachardiaephagus in Malaysia free of pathogens and hyperparasites, to import this population under permit to Christmas Island, to establish and expand this population in a mass‐ rearing facility on Christmas Island have all been identified. An agreed protocol for the rearing and release of biological control agents, and pre‐ and post‐release monitoring for their efficacy in controlling target scale insects and reducing densities and impacts of the invasive yellow crazy ant Anoplolepis gracilipes has also been prepared.

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Introduction Background to the Program

Biology and Impacts of the Yellow Crazy Ant on Christmas Island. Anoplolepis gracilipes (the yellow crazy ant, YCA) is a ‘tramp’ species that has become invasive throughout the tropics (Wetterer 2005). The YCA is listed by the IUCN as one of the world’s 100 worst invasive species (Lowe et al. 2000), and was accidentally introduced to Christmas Island between 1915 and 1934 (O’Dowd et al. 1999). Because of its negative impacts on species, interactions and ecosystem processes, YCA is recognised as the most significant and pervasive threatening process affecting biodiversity on Christmas Island, reflected by the listing of the Loss of biodiversity and ecosystem integrity following invasion by the Yellow Crazy Ant on Christmas Island, Indian Ocean as a Key Threatening Process under the EPBC Act 1999, and as identified in Threat abatement plan to reduce the impacts of tramp ants on biodiversity in Australia and its territories (Commonwealth of Australia 2006). The control of this ant features prominently in many Recovery Plans for EPBC‐Listed species on Christmas Island, and has been the focus of natural resource management activities on the island for more than a decade at the cost of millions of dollars. The attribute that makes the YCA so problematic on Christmas Island and elsewhere is its capacity to form high‐density, expansive ‘supercolonies’. Many tramp ant species form supercolonies, often defined using a combination of criteria including genetic relatedness, intraspecific behavioural interactions, and ant abundance. For example, the invasive argentine ant Linepithema humile is genetically uniform across most of its European range and is regarded as a single supercolony (Giraud et al. 2002), while intercontinental behavioural assays and analyses of cuticular hydrocarbons suggest this species may form a single supercolony spanning Australia, Europe, North America and Japan (Sunamura et al. 2009; Suhr et al. 2010). Although two distinct genotypes of YCA occur on Christmas Island, these co‐occur at very small spatial scales (Thomas et al. 2010) and behavioural assays pairing individual ants from opposite ends of the island suggest that the population on Christmas Island behaves as a single supercolony (Abbott 2005). Nevertheless, YCA supercolonies on Christmas Island have always been defined in terms of very high ant densities. YCA occurred at many locations across the island in very low abundance with no obvious impact on biodiversity, but in 1989, and then again in late 1997, YCA was discovered in several locations at extremely high densities sufficient to extirpate local populations of the abundant red land crab Gecarcoidea natalis (O’Dowd et al. 1999, 2003). The red crab is a keystone species in rainforest on the island that controls patterns seedling recruitment and rates of litter breakdown and nutrient cycling (Green et al. 1997, 1999, 2008). Since then, the density at which YCA kills land crabs leading to understorey transformation has become the operational definition of ‘supercolony’ on Christmas Island. On the ground, the density of YCA in supercolonies can be extraordinary – up to several thousand per square meter (Abbott 2005). A systematic, island‐wide survey in 2001 found multiple supercolonies ranging in area from tens to hundreds of hectares, totalling c. 2500 ha, or 25% of rainforest on the island (Green et al. 2004; Green and O’Dowd 2009). Supercolonies have continued to form and reform; upwards of 5000 ha of rainforest have been treated with toxic bait to 2012. The key trait that has allowed YCA to form high‐density supercolonies is its ability to form mutualistic associations with honeydew‐producing hemipterans, principally scale insects (O’Dowd et al. 2003; Abbott and Green 2007). The scale insects suck sap from trees and secrete carbohydrate‐rich honeydew on which the ants feed. The ants provide sanitation services for the scale insects, removing honeydew that might otherwise build up and kill them either through asphyxiation or the growth of

5 sooty moulds, but the ants may also provide limited protection for the scale insects from generalist natural enemies. Supercolony‐level densities of YCA and outbreak‐densities of several species of scale insects invariably co‐occur (Abbott 2004), and in supercolonies high densities of ants can typically be seen ascending the boles of most trees to tend scale insects in the canopy. The gasters of descending ants are swollen with carbohydrate‐rich honeydew that they take back to the nest to share with non‐ forager conspecifics. Site‐scale, manipulative experiments on Christmas Island have demonstrated a causal link between co‐occurring high densities and ants and scale; the exclusion of ants using toxic bait leads to a dramatic decline in scale abundance (Abbott and Green 2007). Part of the current project was to determine if this link is bi‐directional, that is, if the exclusion of scale insects from the ants leads to a dramatic decline in ant density (see Project 1c, below). On Christmas Island YCA interacts with several species of honeydew‐secreting scale insects including Tachardina aurantiaca, the principal target of the biocontrol program (see below). The mutualism between YCA and scale insects has manifold negative impacts on species abundances, interactions among species, and forest structure. At the core of these impacts is the devastating effect of YCA on land crabs, especially red crabs. YCA sprays formic acid as a weapon both to subdue prey and in self‐defence, and although the amount sprayed by individual ants is tiny, at supercolony densities the overall effect is overwhelming. YCA supercolonies reduce formerly high densities of red crabs (averaging c. 0.5 – 1.0 crabs m‐2) to nil, deregulating seedling recruitment and litter dynamics and resulting in a thick, diverse understorey of seedlings and saplings with an almost permanent layer of leaf litter (O’Dowd et al. 2003). In forest dominated by red crabs, the understorey is sparse and dominated by a few crab‐resistant species, and the forest floor is almost devoid of litter for much of the year (Green et al. 1997, 1999, 2008). These impacts are widespread. Based on the spatial extent of supercolony formation over the last 12 years, it is likely that YCA has extirpated at least 20 million red land crabs, or about 30% of the total population in areas where they have formed supercolonies. YCA has also caused declines in the density of red land crabs at sites where high‐density supercolonies have never formed. About half of the red crab population migrates to the coast each year to complete breeding activities, and many YCA supercolonies have formed across traditional crab migration routes. Thus, significant numbers of migrating red crabs have been killed en route to the coast over many years, never to return to their former home ranges. As a result, some areas of rainforest are practically devoid of red crabs even though YCA supercolonies have never occurred there, and the same processes of understorey transformation are in train there too. It is hard to gauge the severity and extent of this “ghosting” effect because pre‐YCA invasion data on red crab densities are sparse. It is likely to be significant; Green et al. (2011) estimated that around 25% of rainforest may have been ghosted at some time in the last decade. The direct and indirect (ghosting) impacts of YCA supercolony formation have been so widespread since the late 1990s that just 28% of rainforest could still be considered as “intact” (i.e. no YCA supercolony formation and unaffected by ghosting) by 2007 (Green et al. 2011). In YCA supercolonies, scale insects themselves can have large negative impacts on their host plants. Especially vulnerable is the Tahitian Chestnut Inocarpus fagifer, a widespread canopy tree that hosts very high densities of Tachardina on its outer twigs and leaves. In supercolonies, seedlings, saplings and small trees all suffer extremely high mortality, and the canopies of large trees are much reduced through the dieback of fine twigs and branches (Green et al. 2001, O’Dowd et al. 2003, P. Green and D. O’Dowd, unpublished results). There is also evidence that fruit production is reduced in supercolonies. Excess honeydew that YCA does not harvest, or which the scale insects deliberately flick off, settles on

6 leaves of all plant species and is colonised by sooty moulds, which probably interferes with photosynthesis and growth. YCA may affect many of the island’s bird species through direct interference and through altered resource availability and habitat structure (Davis et al. 2008). The Christmas Island Emerald Dove Chalcophaps indica natalis is 9‐14 times less abundant in ant‐invaded forest, and because it forages on the forest floor, is probably vulnerable to direct predation by YCA. The nesting success and density of juvenile Christmas Island Thrushes Turdus poliocephalus erythropleurus is lower in supercolonies, where they also show altered foraging and reproductive behaviours. Furthermore, these birds alter their choice of tree species in which to build nests, with lower frequency on tree species that typically experience high densities of scale insects and ants. The density of foraging Christmas Island white‐eye Zosterops natalis is higher in supercolonies, perhaps because scale insects (as prey) are more common there. It is possible that impacts of YCA on thrushes and white‐eyes affect frugivory and seed dispersal on the island; assays with both real and model fruit showed handling rates to be more than two‐fold lower in YCA supercolonies, and manipulative experiments showed this to be a direct consequence of the presence of ants (Davis et al. 2009). There is no evidence that YCA supercolony formation significantly affects the density of nesting success of Abbott’s Booby Papasula abbotti (P. Green, unpublished data), while the impact of YCA on other seabirds and on other land birds such as the goshawk and owl are unknown. Several endemic vertebrate species, including the pipistrelle bat Pipistrellus murrayi and endemic reptiles have experienced precipitous declines over recent years, but the causes of the declines are enigmatic. In the case of the pipistrelle it is possible that YCA supercolony formation has contributed to the decline, either directly through predation of bats at roost sites or indirectly by eliminating red crabs and facilitating the expansion of predators such as giant centipedes Scolopendra subspinipes, wolf snakes Lycodon aulicus, cats and rats (Schulz and Lumsden 2004, Lumsden et al. 2007, Beeton et al. 2010). However, the decline of the pipistrelle was well in train before the rise of YCA supercolonies in the late 1990s. The endemic reptiles were similarly in decline long before YCA supercolonies became common, and the role of YCA in their decline is also uncertain (Smith et al. 2012). In addition to impacts on species of concern, supercolony formation by YCA has also led to an altered web of species interactions that facilitates the entry, spread, and abundance of other invasive species. The best example of this is the entry and spread of the giant African landsnail Achatina fulica (GALS) in rainforest on the island. GALS was introduced to the island in the 1940s, and despite being a notoriously invasive species (Lowe et al. 2000), its distribution was for many decades limited to settled areas, abandoned mining fields and roadsides. Experiments showed that predation by red crabs excluded this invader from establishing in rainforest (Lake and O’Dowd 1991), but the extirpation of red crabs in YCA supercolonies, coupled with the ability of GALS to coexist alongside YCA in supercolonies, has permitted this snail to establish high densities in rainforest at many locations across the island (Green et al. 2011). The facilitation of GALS by YCA could be due to the creation of enemy‐free space, augmented understorey resources, or both. The rise of YCA supercolonies and the extirpation of red crabs have also affected the invasion dynamics of other non‐native organisms. These effects encompass both inhibition and facilitation for a range of non‐native ant and snail species (O’Dowd and Green 2010; P. Green and L. O’Loughlin, unpublished results), while invasion by several weeds including Capsicum frutescens, Carica papaya, Cordia curassavica, and Muntingia calabura appears to be facilitated in areas affected by YCA supercolony formation (P. Green and D. O’Dowd, personal observations). 7

Management. Given all of the above, supercolony formation by YCA is considered a major and on‐ going threat to biodiversity values on Christmas Island. To date, the management of YCA has depended on surveillance, monitoring, and control using toxic bait (Green and O’Dowd 2009, Boland et al. 2011). New supercolonies continue to form, and there is concern for the sustainability of this program in terms of its expense, non‐target impacts, and the resources it diverts from other conservation programs (Beeton et al. 2010). Further, this program can only ever be reactive, and it has not been able to find an effective solution to the difficult issue of the management of incipient supercolonies, where YCA density is high enough for them to monopolize the toxic bait, making the risk of non‐target impacts too high to justify the treatment of these areas. There is widespread agreement that the development of a more cost‐effective, sustainable alternative to the use of toxic bait is needed to manage the YCA invasion on Christmas Island (Beeton et al. 2010). Ten years ago, O’Dowd and Green (2003) suggested that long‐term, sustainable suppression of YCA supercolonies could be achieved using biological control agents. Classical biological control works on the principle that in their area of origin, populations of native species are kept in check by their natural enemies (predators, parasites or pathogens). There is a large body of literature demonstrating that in many cases, species introduced outside of their native ranges become invasive because they have effectively left their enemies behind. This is known as the “Enemies Release Hypothesis”. The principle of classical biological control then is the converse: to re‐establish population control over invasive species by first identifying and then importing a natural enemy – a biological control agent – from within the native range of the target organism. In all instances, this now involves selection and testing of biological control agents to verify narrow host ranges that minimize the risk of non‐target impacts in the area of introduction.

Indirect Biocontrol for YCA on Christmas Island. Despite the diversity and significant ecological and economic impacts of invasive ants worldwide, they have proved to be an especially difficult target group for biological control. Although a program for the biocontrol of the Red Imported Fire Ant (Solenopsis invicta) using a parasitic fly and a protozoan disease as agents is currently under development in the southeastern United States, no species of ants have yet been successfully controlled in the field using biological control agents and principles. Instead, O’Dowd and Green (2003) proposed a novel solution for managing the YCA invasion; rather than targeting the ant itself, biological control could target the key mutualist species (honeydew‐producing scale insects) that are likely to play a significant role in sustaining YCA supercolonies at very high and ecologically damaging densities. Long‐ term, sustainable suppression of YCA supercolonies could be achieved through the introduction of a biological control agent that would indirectly affect YCA by reducing carbohydrate supply provided by honeydew‐producing scale insects, a key resource implicated in supercolony dynamics.

The Biocontrol Project 2009 – 2013. Funding for a four‐year program of research and development to progress the concept and feasibility of indirect biocontrol was allocated in the Australian Federal Budget 2007/08, as part of a broader, 10‐year plan for the control of YCA on Christmas Island. We proposed a research project with three overarching questions: 1. Do YCA depend on honeydew‐producing scale insects? 2. Which species of honeydew‐secreting scale insects occur on Christmas Island, and are appropriate natural enemies for scale insects available for introduction? 3. What regulatory frameworks govern the implementation of a biological control program on Christmas Island, an external territory of Australia?

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Two Postdoctoral Fellows were employed to perform most of the research under Questions 1 & 2. Dr. Sarah Wittman was recruited from the University of Vermont in the United States, with a project brief to establish the dependence of YCA on honeydew‐producing scale insects. She arrived on Christmas Island in November 2009, and finished in December 2012. She is currently back in the United States working on the preparation of manuscripts for publication arising from her work. Dr. Gabor Neumann was recruited from the USDA‐ARS in Hilo, Hawaii as a biocontrol practitioner, with a project brief to investigate the scale insects of Christmas Island, to describe their local suite of natural enemies, and to search for suitable biocontrol agents in Southeast Asia. His tenure on this contract expires in July 2013.

The biocontrol project was overseen by an independent steering committee, the Christmas Island Crazy Ant Scientific Advisory Panel (CASAP), which comprises scientists, managers, and policymakers. CASAP provided expert advice and reviewed progress through panel meetings and evaluation of interim and annual reports.

Structure of the Final Report. The broad research questions (above) were refined into three “Research Projects” and 13 subprojects, that were listed as Services in the Schedule to the Contract between the Director of National Parks and La Trobe University, signed on 29 January 2009. Below, we report against these subprojects as they are laid out in the Contract Schedule, and provide an introductory rationale for each using text (in italics) copied from the original Schedule.

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Research Project 1. YCA dependence on honeydew Numerical dominance and impacts of invasive ants may be sustained by their association with honeydew‐secreting Hemiptera. For example, ~1/2 of the energy supply of the red imported fire ant is obtained from honeydew secreted by Hemiptera and ecologically dominant ants in the rainforest canopy are highly dependent on honeydew. How dependent is the YCA on honeydew obtained from scale insects and is honeydew critical to population size? Research outlined below is directed at estimating the use and sources of honeydew by the YCA on Christmas Island, the importance of carbohydrates in its diet, and the role of honeydew in sustaining high populations.

Project 1a. Honeydew use by YCA Mutualisms between invasive ants and Hemiptera may drive positive population growth leading to numerical dominance of ants. This project will give a quantitative picture of honeydew use by the YCA and focus on the relative importance of the lac scale Tachardina aurantiaca and soft scale Coccus spp. as sources of honeydew. Knowledge of the importance of honeydew derived from lac scale is critical since in the first instance they are the proposed target of biological control. Research Findings 2009‐2013 The original intention of this project to combine data on per capita honeydew production by the most important (common) honeydew‐producing scale insects, analyses of the quality of honeydew (sugars and their concentrations), and estimates of scale abundance and YCA visitation rates to estimate empirically the ‘honeydew economy’ of YCA supercolonies, and the relative contribution of Tachardina to that economy. However, collecting sufficient honeydew for analyses using glass microcapillary tubes proved technically very challenging, there were also issues (packaging, quarantine) of transporting fresh samples to our preferred analytical laboratory in Melbourne. The logistics of collecting spatially and temporally replicated data on YCA visitation rates to scale insects using video recordings was also extremely challenging. Instead, we devised an indirect measure of the honeydew economy, called the Site Honeydew Index (SHI). The SHI is a relative estimate of the total volume of honeydew that could be produced at a site. It is a compound measure that considers the capacity of different plant species to host either Tachardina aurantiaca, or coccoid honeydew‐producing species of soft scale insects, together with tree size and tree abundance. The basis of the SHI is canopy surface area, generated for each tree using published allometric equations that predict crown dimensions from stem diameter (Poorter and Bongers 2006). Presumably, large trees have the capacity to host more scale insects, and canopy surface area is preferable to canopy volume because scale insects live on leaves and thin twigs of the outer canopy. This estimate is then multiplied by the average abundance of scale insects per unit length of stem, using host species‐specific data for both Tachardina and soft scales (data from Table 4.3 in Abbott 2004). The SHI is the sum of these products, divided by 10000 for convenience. Thus, the SHI is the sum of the contributions from individual trees for both Tachardina and soft scales, weighted by tree size (canopy surface area) and species identity. SHIs were calculated for 10 50 m x 50 m plots in YCA supercolonies, on which all trees ≥ 5 cm dbh were enumerated in June‐October 2000 (P. Green, unpublished data). The SHI varied 1.6‐fold (from 78 to 125) suggesting that the capacity of forest stands to support YCA supercolonies varies considerably. Tachardina is estimated to contribute a large fraction of the 10 honeydew economy at forest sites with YCA supercolonies (mean = 70%, range 46‐86%; blue symbols in Fig. 1). This assumes per capita parity in the quantity and quality of honeydew produced by Tachardina and the coccoid soft scales species. However, adult female Tachardina are much larger than the adult females of the coccoid species, and its reasonable to suppose that per capita honeydew production is much higher in Tachardina. Assuming a three‐fold higher rate of honeydew production, Tachardina may contribute a mean of 87% to the SHI (range 72 – 95%; red symbols in Fig. 1). Successful biological control of the yellow lac scale Tachardina aurantiaca could remove a large fraction of honeydew available to the yellow crazy ant. One caveat to this conclusion is that the balance between the contributions made by Tachardina and the coccoid soft scales to the total honeydew economy may have declined over time at some sites where supercolonies have reformed after baiting. Inocarpus fagifer is a key host species for Tachardina in YCA supercolonies; just this single species contributed an average of 50% to the Tachardina component of the SHI in 2000. The species is so heavily utilised by Tachardina and YCA that significant tree mortality occurred in supercolonies between 2000 and 2002 on the 10 50 x 50 m tree census plots (P. Green, unpublished data). Although these supercolonies were destroyed by the aerial baiting campaign of 2002 (Green and O’Dowd 2009) and YCA supercolonies have not reformed on these sites since (D. Maple, unpublished data), a targeted recensus of Inocarpus on 8 plots in 2012 showed some further dramatic declines, possibly caused by Tachardina tending by the ant Camponotus sp. in the decade after baiting. The death of many mid‐ to large‐sized Inocarpus trees has resulted in a decline of that species’ contribution to the SHI by an average of 50% (range 11 – 91%; Fig. 2). The implication of this finding is that where contemporary YCA supercolonies have reappeared at former but baited YCA supercolony sites, the new supercolonies may now be supported to a greater extent by soft scales, and because of that, the introduction of a parasitoid against Tachardina may be less effective in those locations. The extent of this issue could be estimated from the Island‐Wide Survey data by determining the proportion of supercolonies that reform, and the proportion of time supercolonies occupied each site.

Key Research Outcome: Successful biological control of the yellow lac scale Tachardina aurantiaca could remove a large fraction (an average of 70% to an average of 87%, depending on assumptions) of honeydew available to the yellow crazy ant. However, there is considerable site‐to‐site variation in the likely contribution of T. aurantiaca to the total honeydew economy, and it is not certain that targeting this species alone would provide adequate indirect control for the YCA in all supercolonies. The most prudent course of action is to target the entire assemblage of honeydew‐producing scale insects through the introduction of a biological control agent for T. aurantiaca, complemented by the use of parasitoids already present on Christmas Island to target the coccoid soft scales.

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Project 1b. Stable Isotope Analysis Analyses of Nitrogen (N) isotope ratios show that ecologically dominant ants and many invasive ants obtain little N through predation and scavenging, but feed primarily as herbivores, deriving carbohydrate through exudates produced by Hemiptera, primarily scale insects. This project will use this widely accepted technique to determine N and C stable isotope ratios of the YCA, other ant species, and known primary producers, herbivores, and predators to characterize how much of the dietary requirements of the YCA is derived from plant‐derived carbohydrates and how this changes with invasion dynamics. Research Findings 2009‐2013 Honeydew from scale insects is essentially plant sap that has passed through the bodies of scale insects, so when feeding on honeydew YCA are essentially acting as herbivores. If YCA at supercolony densities derive most of their dietary intake from scale insects, then stable isotope analysis of their body parts (δ15N) should indicate a mainly herbivorous diet, while YCA at non‐supercolony densities should show a less herbivorous diet. We estimated the change in trophic position of YCA at four sites (Island Wide Survey points) as the abundance of ants declined from supercolony to non‐supercolony densities. YCA workers were sampled 6 to 8 times over a 16‐month period (April 2010 – September 2011). Trophic position was calculated from mixing models that considered site‐specific stable isotope ratios of known plants, herbivores (stick insects), and carnivores (spiders). All samples were analysed by a commercial company, Natural Isotopes, in Perth, Western Australia. Stable isotope analyses showed that trophic position increased (i.e. YCA became more carnivorous) as population densities decreased over 16 months (Site 206, R2 = 0.75, P = 0.025; Site 318, R2 = 0.67, P = 0.025; Site 403, R2 = 0.58, P = 0.046, Site 582, R2 = 0.72, P = 0.033)(Fig. 3). This result suggests that a waning carbohydrate supply, as indicated by an upward shift in the trophic position of YCA, is associated with supercolony decline. A series of YCA collected over two years from 10 supercolonies in 2000‐2002 were also analyzed for δ15N after storage in ethanol for over a decade. Unlike the four sites above, δ15N values and YCA abundance were not negatively correlated at three supercolonies that declined to zero over the period. However, as expected, δ15N values for the other seven supercolonies with consistently high YCA densities did not vary over time.

Key Research Outcome: Stable isotope analyses of YCA workers, plants, herbivores and predators collected from four declining supercolonies in 2010‐2011 indicate that at supercolony densities, a substantial fraction of YCA dietary intake is plant‐derived. This is consistent with the idea that YCA supercolonies depend heavily on honeydew derived from scale insects for a large fraction of colony food and energy requirements, and provides support for the idea that indirect control over YCA supercolonies could be achieved by targeting honeydew‐producing scale insects for biological control.

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Project 1c. Scale Insect Removal Experiment Although Projects 1a and 1b above will indicate the importance of honeydew in the diet of YCA and the relative contribution by scale insect type, large‐scale field experiments are needed to establish the influence of scale insects on population dynamics of the YCA. This experiment uses the systemic insecticide imidacloprid to remove scale insects in YCA supercolonies and follows ant population dynamics. Research Findings 2009‐2013 The original intention of the is experiment was to use the systemic insecticide imidacloprid to remove scale insects from an experimental plot, and to monitor ant activity before and after to determine the dependency of YCA at supercolony densities on scale insects. This was essentially a proof‐of‐concept experiment for indirect biocontrol. The intent was sow the treatment plot with tablets containing imidacloprid, which would be taken up by the root systems of trees and transmitted to scale insects, removing them from the canopy. This would mimic the effect achieved through the introduction of a biological control agent for scale insects on the honeydew supply to YCA. The experiment was not carried out as planned. Although imidacloprid is routinely used in single species Eucalyptus plantations to control herbivorous insect pests, it has not been used in natural forests where plant species diversity, and a range of size classes of trees from sapling to canopy giants, poses significant challenges for achieving consistent uptake of the toxin across all individual trees. In addition, analyses of honeydew would have been mandatory to test the possibility that any decline in YCA density on the treatment plot was more likely due to scale insect death and a reduction in the honeydew supply, rather than a toxic effect due to the presence of imidacloprid or it metabolites in honeydew. As pointed out above, collecting and preparing honeydew for analysis was challenging in itself. Instead, we used a physical barrier applied to tree trunks to prevent YCA from accessing their honeydew supply in tree canopies. The experiment was a Before‐After‐Control‐Impact (BACI) design on two forest plots in a YCA supercolony abutting the Winifred Beach Track. There were two plots, each 40 m x 40 m, and one was designated as the control plot, the other as the treatment plot. On the treatment plot, tree bands were made by winding Gladwrap™ around the boles of all trees >2 cm DBH. Bands blocked YCA traffic flow to and from the forest floor, resulting in “log jams” of downward moving ants, most replete with honeydew, above the bands (Fig. 4a). Throughout the experiment these stranded ants were returned to the forest floor by brushing them gently off each bole, so that any decline in YCA abundance on the ground could not be attributed to the retention of these ants on trees. Repeated application of Mr Sheen™ spray‐on furniture polish to each band over the experiment greatly increased the effectiveness of the barriers. No bands were applied to trees on the control plot. The measured response variables were YCA trunk traffic and YCA ground activity, both measured in the inner 20 x 20 m core of each plot. YCA trunk traffic was estimated by counts per 30 s in a 10 x 10 cm quadrat on each of 10 trees, while ground activity was monitored using ant counts per 30 s on one quadrant on each of twelve 20 x 20 cm cards. (Fig. 5a). Both variables were monitored at 3‐4 day intervals, 9 times before and after applying tree bands, during the dry season in 2012. Results were analyzed as a one‐way repeated measures ANOVA, using ant exclusion from the forest canopy as the main factor and time as the repeated measures factor. In this design, the time x treatment interaction is the key term, with a significant treatment difference after, but not before treatment application.

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Bands effectively excluded YCA from the canopy, resulting in a precipitous decline and the virtual elimination of YCA traffic on tree boles 4 weeks after the tree bands were in place (Fig. 4b; Treatment x

Time interaction, F1,32 = 90.198, P = 0.000). YCA abundance on the ground fell and diverged markedly from the control plot two weeks after tree bands were placed (Fig. 5b; Treatment x Time interaction,

F1,32 = 37.604, P = 0.000). YCA abundance declined ~3‐fold compared to average pre‐exclusion values, and was ~5‐fold lower than on the control plot 4 weeks. If card counts are converted to YCA densities

(using the regression y = 15.694x – 21.612; Abbott 2005), YCA densities on the forest floor fell from ~400 ‐2 ‐2 m before to ~140 m after exclusion from the canopy, and when the experiment was terminated were ‐2 ‐2 ~600 m on the control plot compared to ~100 m on the exclusion plot. We attribute the decline in YCA density on the ground to their exclusion from honeydew resources from scale insects in the canopy. Two alternative explanations are not likely. First, plot disturbance when setting up the tree bands on the treatment plot could have affected YCA activity on the ground but would be unlikely to explain the magnitude of change in YCA abundance after bands were in place. Second, this experiment excluded YCA from all canopy resources, not just honeydew, and its possible that it was exclusion from other food sources, such as invertebrate prey, that caused the observed decline in density on the ground. In anticipation of this, video records were made of samples of ants descending tree trunks to determine the proportion with distended shiny gasters (indicative of honeydew foraging), versus the percentage of workers carrying prey items. These records are still being processed, but observations indicate most workers were foraging for honeydew, and < 1% were carrying prey.

Key Research Outcome: The exclusion of YCA from access to scale insects at a large experimental field site caused YCA activity on the ground to decline 5‐fold within 4 weeks, compared to pre‐treatment levels. This large field experiment validates the key concept of indirect biological control for YCA on Christmas Island; exclusion of honeydew‐producing scale insects from YCA caused a significant and substantial reduction in YCA abundance on the ground.

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Project 1d. Carbohydrate supply and YCA growth and behaviour The importance of simple carbohydrates in the population growth and behaviour of the yellow crazy ant (YCA) will be assessed by varying carbohydrate supply to laboratory colonies and measuring brood production, worker survival, and aggression. Research Findings 2009‐2013 This experiment consisted of nine treatments that varied the absolute amount, but not the concentration, of sugars available to YCA colonies. Eighteen YCA nests were set up in plastic containers sitting in water traps to contain the ants. Each nest initially contained 2 queens and 200 workers, and they had unlimited access to water and protein (thawed crickets) for the duration of the experiment. Two nests were assigned to each of nine sugar treatments, 0, 10, 20, 40, 80, 160, 320, 640, and 1280 µl, delivered as 13% honey water every 3‐4 days for two months. In the last week, novel objects (a small frame of skewers) were added to track YCA exploratory behaviour, and behavioural assays against the big‐headed ant Pheidole megacephala were conducted to assess the impact of sugar supply on interspecific aggression of YCA. In these aggression assays, three YCAs and three Pheidole were placed in a 6 cm diameter vial with fluon‐coated sides for 10 minutes. The time to the first spray of formic acid by each YCA, the total number of sprays by each YCA, and the total number of P. megacephala dead after 10 minutes was recorded. Performance and foraging behaviour of yellow crazy ants depend on sugar supply. Per capita recruitment to sugar indicated that a smaller fraction of colony workers was needed to collect sugar with increasing sugar supply (Fig. 6a). Colony performance, as measured by production of workers and males (Fig. 6b) increased with sugar availability (P = 0.034) whereas per capita death rate decreased with increasing sugar availability (Fig. 6c). Per capita encounter rate of YCA with novel, non‐food objects placed in the nest, a measure of foraging tempo, also increased with sugar supply (Fig. 6d). Aggressive behaviours in the yellow crazy ant increased with sugar supply. YCA with access to more sugar sprayed P. megacephala with formic acid sooner and more often (Fig. 7a; P = 0.006), and killed more P. megacephala in 3:3 interaction trials (Fig. 7b).

Key Research Outcome: Dynamics and behaviour of YCA in laboratory colonies depended on carbohydrate supply. When sugar supply was elevated, reproductive output by queens increased, death rates of workers decreased, foraging tempo quickened, and interspecific aggression intensified. These results suggest that sugar supply, through honeydew supplied from scale insects, plays an important role in YCA supercolony dynamics, foraging efficiency, and interspecific aggression.

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Research Project 2. Scale insects and natural enemies For indirect biological control of the YCA to be feasible, appropriate natural enemies must be available. The most likely source of specialist natural enemies is in association with the target scale insect in its area of origin. Our co‐operator (Dr. R. Pemberton, USDA‐ARS Ft Lauderdale FL) is already well advanced in development of biological control of the lac scale Paratachardina sp. nov. and recently determined SEA as its area of origin. Further, he agreed to search for and collect Tachardina aurantiaca in Thailand and Malaysia for us from which they have now reared several species of hymenopteran parasitoids that are currently being described. Research outlined below is directed at building on these preliminary results to locate and evaluate potential biological control agents for T. aurantiaca in SEA, defining the scale insect fauna of Christmas Island, determining any natural enemies of scale insects already on Christmas Island, and establishing a pre‐release monitoring program for T. aurantiaca.

Projects 2a and 2c. Natural enemies survey, parasitization rates of lac scale insects (Tachardina aurantiaca and Paratachardina pseudolobata) and parasitoid behaviour in area of origin (Southeast Asia) and on Christmas Island 2a Collection of scale insect material to rear enemies and identification of candidate parasitoids for consideration in biological control. Species descriptions, biology and behaviours of suitable candidates as biological control agents. Separation of parasitoids into wanted primary parasitoids and undesirable hyperparasitoids. 2c Identification of existing natural enemies (if any), and predation and parasitism rates on key scale insects on Christmas Island. This will identify if significant natural enemies of scale insects are already present on the island. Research Findings 2009‐2013 This part of the research very quickly focused on Tachardina aurantiaca as the key Kerriidae honeydew producer on Christmas Island. Although Paratachardina pseudolobata produces honeydew, females flick droplets away from their bodies (Howard et al. 2010) making it unavailable to foraging YCA. Below, we present information on the target T. aurantiaca including its geographic range in Southeast Asia, its life cycle, its natural enemies and incidence of parasitism in Southeast Asia and on Christmas Island, and a description of the biology of its candidate biological control agent, Tachardiaephagus somervillei. We conclude this section with a short description of the suite of parasitoids of soft scales found on Christmas Island. Distribution of T. aurantiaca in Southeast Asia. We located populations of T. aurantiaca across a 1900‐ km east‐west distribution in Sundaland, its putative area of origin (Fig. 8). This region is the part of Southeast Asian continental shelf that was exposed during the last ice age, encompassing Peninsular Malaysia, Borneo, Java, and Sumatra). Much of the search for T. aurantiaca in Malaysia was targeted, using a highly suitable and widespread host plant, Milletia pinnata, as a focus. Search effort totalled 27 days In Malaysian Borneo, and 5 days were spent searching in Singapore. Live aggregates of yellow lac scale (yellow circles in Fig. 8) were located on Penang Island, and in Klang (Selangor) and Kepong

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(Selangor) in Peninsular Malaysia. In Singapore, T. aurantiaca was found on the campus of the National University of Singapore. In Sarawak, live aggregates were discovered around Santubong and Kuching (two sites ‐ Kampung Istana and Kampung Boyan). In Sabah, live aggregates were found in the Sandakan area and Sepilok. Dead aggregates (not shown above) were found in Melaka, peninsular Malaysia and in Temon, 190 km south of Kota Kinabalu, Sabah. Ants tended T. aurantiaca at all sites and collected honeydew (Penang – Crematogaster sp.; Klang – Oecophylla smaragdina; Kepong – Dolichoderus sp.; Kampung Istana – Anoplolepis gracilipes; Kampung Boyan – Oecophylla smaragdina; Singapore, Sandakan and Sepilok – Anoplolepis gracilipes). Assemblage of natural enemies of T. aurantiaca in Southeast Asia and on Christmas Island. On Christmas Island, search focused on seven areas (see Figure 12) and examined over 11,000 females and 2000 males of the yellow lac scale. In Malaysia, targeted search for T. aurantiaca on known host plants (e.g., Milletia pinnata, Acacia mangium x A. auriculiformis) was frequently used to locate T. aurantiaca. Because the yellow lac scale is so rare across Malaysia, many fewer individuals were inspected for parasitization. To determine natural enemies, host plant twigs with aggregates of T. aurantiaca were first inspected, and then isolated so that emerging parasitoids could be collected for later identification. Female T. aurantiaca, either individually or in aggregates, were inspected for parasitoid exit holes to estimate parasitization rates. Individual females were also isolated to collect emergent parasitoids and dissected to determine overall rates of parasitization. The assemblage of natural enemies of T. aurantiaca is much more diverse in the area of origin that in its introduced range on Christmas Island (Table 1). Using a combination of historical records (Noyes 2012) and direct field surveys, there are six primary parasitoids that use T. aurantiaca as a host in Malaysia, but only one on Christmas Island. Furthermore, the one parasitoid on Christmas Island, Marietta leopardina, can only successfully attack males of T. aurantiaca. Females of this species attempt to lay eggs inside females of Tachardina, but the test is seemingly too tough for the ovipositor to penetrate. In addition to parasitoids, two lepidopteran predators of female T. aurantiaca were also been found in both the native and introduced ranges, but on Christmas Island these are extremely rare. Two hyperparasitoids, Promuscidea unfasciativentris and Aprostocetus purpureus, were isolated from T. aurantiaca in Malaysia, but not on Christmas Island (Table 1). Parasitism of T. aurantiaca in its native and introduced ranges. In Southeast Asia, T. aurantiaca is rare and evidence of female parasitization was frequently seen in its native range in SE Asia (Fig. 9). In contrast, in its introduced range on Christmas Island, T. aurantiaca is superabundant and there was no evidence of female parasitization (Table 2). In Southeast Asia, the rate of parasitism was high, varying from a mean of 29% to 81% at different sites. The rates in Table 2, based on the incidence of emergence holes, almost certainly underestimate actual parasitization rates because parasitized females from which parasitoids have not yet emerged would not be included. To investigate this, parasitisation rates of 5 additional aggregates in Sarawak were assessed by counting emergence holes, and then by dissecting the females to count unemerged parasitoid larvae. Parasitization was estimated as 44.6 ± 9.6% (mean ± SE) by emergence holes, but as 61.4 ± 7.7% when females with unemerged larvae were added. As a further example, a small aggregate near Kuching that showed no visible sign of parasitization (i.e. no exit holes) was found to be 100% parasitised after dissections. Tachardiaephagus somervillei parasitized T. aurantiaca across all sites examined (Fig. 9a). Superparasitism, where more than one progeny is produced per host individual, is frequent. The mean number of T. somervillei emerging from each parasitized female T. aurantiaca was 2.1 (range 1‐ 4, N = 30) and up to 5 emergences occurred from each female at sites in Peninsular Malaysia. Coccophagus 17 tschirchii and C. euxanthodes were also isolated from T. aurantiaca (Table 1), but these species were uncommon and found only in Peninsular Malaysia (Fig. 9b). In contrast to the widespread parasitism of T. aurantiaca in Southeast Asia, parasitism of females was never observed on Christmas Island. More than 11,000 females from multiple sites (Table 2) were collected and inspected under magnification over two years to determine the presence of any parasitoid emergence holes. Single exit holes were observed in some male T. aurantiaca at a few sites. In the laboratory, the primary parasite Marietta leopardina emerged from these males. Parasitization rates were low, ranging from 0 ‐ 10% (N= 558, 751, and 696 males examined on three trees at Hugh’s Dale, Anderson’s Dale, and Sydney’s Dale), but M. leopardina is clearly not an effective biological control agent of female T. aurantiaca on the island. High densities of intact females occured where M. leopardina was present and, on Christmas Island, T. aurantiaca may be parthenogenetic (G. Neumann, unpublished results). Biology, ecology and life cycle of the parasitoid microhymenopteran wasp, Tachardiaepagus somervillei. T. somervillei was first described by Mahdihassan in 1923 as Lissencytus somervilli but in 1928 Ferriere transferred L. somervillei Mahdihassan to Tachardiaephagus and also later downgraded the species somervillei to a variety of T. tachardiae Howard (Hayat et al. 2010). However, Hayat et al. (2010) regarded T. somervillei as a valid species. T. somervillei is known to occur in India, Thailand and Malaysia (Hayat et al. 2010 and references within). During our exploration for natural enemies of Tachardina aurantiaca, T. somervillei was found and its identity was confirmed by Dr. Mohammad Hayat (Table 7) from sites in Selangor, West Malaysia and in southwestern Sarawak, East Malaysia. We also observed this parasitoid in eastern Sabah, East Malaysia but its identity has not yet been confirmed. With its large geographical range (Fig. 8), T. somervillei is a very widespread and frequent natural enemy of T. aurantiaca. Host records for T. somervillei indicate that this parasitoid attacks four species in the genus Kerria (Noyes 2012) and T. aurantiaca (Table 3). The host records sometimes do not specify the species of Tachardina parasitized but the only known species in the genus Tachardina within the recorded geographical range of T. somervillei is T. aurantiaca. The apparently restricted host range of T. somervillei based on historical records is not surprising: all known parasitoids within the genus Tachardiaephagus have been recorded only from lac scales (Kerriidae), with the single exception of a coccid (Ceroplastes eucleae) recorded as a host of T. similis in Africa. This latter host record, however, has been discounted by Prinsloo (1977). While host ranges of the species in the genus Tachardiaephagus have not been assessed in detail, Sharma et al. (2006) mentions T. somervillei (along with T. tachardiae) as “exclusive to lac ecosystem”. Besides taxonomic work, very little research has been done on the biology of Tachardiaephagus spp. even though the species attacks economically important lac scales, such as Kerria lacca (Kerr) and are considered pests in lac‐producing regions (Sharma et al. 2006). Our initial observations in peninsular Malaysia and Sarawak provide basic information on the life cycle and biology of T. somervillei in relation to T. aurantiaca. The basic biology and life cycle is similar to that of other encyrtids that attack scale insects. T. somervillei attacks mature female hosts, but never attacks males. Parasitoid eggs are deposited inside the host’s test (the test is a secretion that covers the scale insect’s body), probably through the anal pore, and the hatching parasitoid larvae consume the host’s body inside the test. The larva pupates after larval development is completed. The adult wasp emerges from the pupa and through a round, smooth‐edged whole which it drills through the test and exits the dead host. Development time from oviposition to adult emergence can vary, but it is usually about 3 weeks. 18

More than one parasitoid may emerge from a single host (superparasitism), and the number of emerging progeny is correlated with host size. In order to investigate the relationship between the number of emerging parasitoid progeny and the size of the female T. aurantiaca host in field conditions (Kuching, Sarawak), a random sample of 50, parasitized female T. aurantiaca were measured (greatest horizontal diameter of test) and the emerging adult parasitoids were counted from each host. There was a significant positive correlation between host size and parasitoid progeny produced (R2 = 0.47, p < 0.001) (Fig. 11). This relationship is probably best explained by the simple fact that larger hosts provide more food resources for more parasitoid larvae to complete development. It is not clear whether this superparasitism is self‐superparasitism (a single female parasitoid ovipositing multiple eggs in a single host) or if it is superparasitism in terms of intraspecific competition (different individual parasitoids ovipositing in the same single host). This positive relationship between host size and production of T. somervillei progeny will be important in captive rearing, release site selection, and ensuring establishment during inoculative releases. The longevity of adult parasitoids and their fecundity can vary greatly depending on diet and the source of nutrients such as sugar from honeydew or nectar (Lee et al. 2004). It is possible that T. somervillei feeds on honeydew secreted by its host. Further, some parasitoids stab their hosts with their ovipositor but do not lay eggs, instead feeding on the hemolymph that leaks out. Such "host feeding" can also be very important in longevity and fecundity (Wheeler 1996). Host feeding, although not yet observed in the field, would be advantageous because it can result in the death of the host without it being parasitized. Natural enemies of soft scales on Christmas Island. Parasitization of soft scale insects (Coccidae) on Christmas Island was very rare during the searches between 2010 and 2012. Signs of parasitoid presence were initially found only in the settled area in the northeast of the Island. The reason for the apparently limited distribution of soft scale parasitoids on Christmas Island is not clear. The 2010 dry season was unusually wet and scale insect populations were very low in general throughout the island. It is possible that parasitoid populations went locally extinct in areas outside of the settled areas and the recolonization of soft scale aggregates simply did not occur at detectable levels in 2011 and 2012. Alternatively, despite their presence in the settled areas, these parasitoids are naturally absent from large parts of the rainforest on Christmas Island due to dispersal limitation. Parasitization was observed in Coccus sp. and Pulvinaria urbicola, a newly recorded invasive soft scale on the island (Neumann et al. 2011). Two parasitoids, Coccophagus ceroplastae (Aphelinidae) and Encyrtus infelix (Encyrtidae) were recovered from Coccus sp., both first recorded in the settled areas. Additionally, two Metaphycus spp. (Encyrtidae) and Coccophagus nr. bivittatus (tentatively identification) were recovered from immature soft scales (most likely P. urbicola) near the 'Boulder' (South Point) on the host plant Pisonia grandis. Coccophagus ceroplastae and E. infelix are both known to attack a variety of soft scale species, including three that are important in YCA supercolonies (Table 4: Coccus hesperidium, Saissetia oleae, S. coffeae). It is possible that these two parasitoid species could be used to control all, or almost all, soft scales at acceptable levels on Christmas Island. For example, C. ceroplastae can control the damaging scale insect P. urbicola under laboratory conditions. Potted Pisonia umbellifera plants were infested with P. urbicola and the scale insects were allowed to reproduce. When the scale insects of the first generation were estimated to be in the second instar of their development, 60 field‐collected C. coccophagus were liberated on the plants in two batches one week apart. Parasitism was observed approximately three weeks later. Parasitization rates were low and not quantified, but the suitability of the host was 19 demonstrated. Approximately 11 weeks after the first parasitoid introduction, two of the host plants had no remaining live scale insects. The other two plants died before the eradication of the scale insect infestation but parasitism rates were high (G. Neumann, unpublished data). The potential of C. nr. bivittatus and the two Metaphycus species is not clear. Study of the potential of these parasitoids will commence in Phase 2 of the biological control project as well as the mass rearing, redistribution, and impact survey of the identified soft scale parasitoids, C. ceroplastae and E. infelix.

Key Research Outcomes: Few natural enemies of the key honeydew‐producing scale insect Tachardina aurantiaca occur on Christmas Island, and they do not regulate its population size. No parasitoids of female T. aurantiaca were found. Conversely, within its native range in Malaysia, T. aurantiaca is rare and patchily distributed, associated with diverse natural enemies, including at least five species of primary parasitoids, and it suffers high parasitization rates – all attributes consistent with population regulation by natural enemies. Importantly, high parasitism rates in Malaysia occurred in the presence of honeydew‐collecting ants, including the yellow crazy ant. Of the primary parasitoids known to attack yellow lac scale from our studies in Malaysia, Tachardiaephagus somervillei (Encyrtidae) is the most promising agent for introduction and release on Christmas Island. All Tachardiaephagus species have a narrow host range and appear to be family specialists, known only to attack the Kerriidae, the family to which the yellow lac scale belongs (Table 3). Our initial studies indicate that (a) T. somervillei attacks T. aurantiaca across 1900‐km range in Peninsular Malaysia and Malaysian Borneo, (b) is the most abundant natural enemy of T. aurantiaca, (c) has a short life cycle compared to its host, (d) exhibits superparasitism (i.e., where multiple progeny emergence from a single host individual); (e) causes high rates of parasitism on T. aurantiaca in the presence of tending ants, including the yellow crazy ant, and (f) can be reared under laboratory conditions. Coccophagus ceroplastae and Encyrtus infelix, both parasitoids of a wide variety of coccid scale insects, are already present on Christmas Island. The three parasitoids T. somervillei, C. ceroplastae and E. infelix could be deployed in combination against the entire assemblage of honeydew‐producing scale insects in YCA supercolonies.

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Project 2b. Scale insect survey on Christmas Island (biology, host range, habitats, natural history, identification) Comprehensive knowledge of the scale insect species present in rainforest and the built environment on Christmas Island. This information will indicate habitats and host plants of T. aurantiaca and other scale insects on Christmas Island. It will provide information on nativeness of the scale insect fauna which will aid determination of a host specificity test list, testing protocols, and other regulatory matters for biological control agents. Research Findings 2009‐2013 Extensive searches for scale insects were carried out over three years 2010‐2013, using five methods. First, surveys were conducted using Timed Searches, in which 30 min was spent looking within a 50‐m radius at each of 151 waypoints from the CINP island‐wide survey (Fig. 12). Searches were replicated three times in each dry season in 2010‐2012. Second, searches were conducted along the entire length of five tracks, a total of 21 km, twice in each of the dry seasons of 2011 and 2012 (Northwest Point Track, 4.7 km; Boulder Track, 6 km; Blowholes Road, 3.4 km; Martin Point Lookout to CINP boundary,

1.9 km; and, Dolly Beach Boardwalk, 1.8 km). Search effort totalled ~405 hours that did not include travel between sites. Third, endemic plant species were a focal point of further search assuming that endemic scale insects would be more likely to be associated with them (Neumann et al. 2007). Fifteen of the 18 species of endemic plant species were located and examined between 2010‐2012. Ten rare and endemic plants species from the CINP database were searched for scale insects at 125 locations. For each common endemic plant species, 30 haphazardly selected individuals were examined each year between 2010‐2012. Fourth, opportunistic searches were made of exotic plants at many locations around the settled areas in the ‘Dog’s Head’ area of the island. Fifth, CINP personnel also aided the search by being aware of scale insects during the island‐wide survey in 2011. Twenty‐one species of scale insects in six families have been found on Christmas Island (Table 4). It is highly probable that these species, all with broad host plant ranges and geographic distributions, were introduced following human settlement on Christmas Island and are non‐native. No endemic scale insects were found.

Key Research Outcomes: No native or endemic scale insect species have been discovered in intensive and extensive searches for scale insect species on Christmas Island. However, 400 hours of search over two years did yield five additional exotic scale insect species previously unknown to the island. Assuming that any prospective biological control agent for Tachardina aurantiaca on Christmas Island would be a narrow family specialist (Kerriidae) and all known scale insect species are non‐native and invasive, the probability of any direct non‐target effects is negligible.

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Project 2d. Genetic and morphological matching between Tachardina aurantiaca and Paratachardina pseudolobata on Christmas Island and Southeast Asia. Determination of species identity and likely area of origin of T. aurantiaca and P. pseudolobata. This is critical for further targeted search for biocontrol candidates. Specimens from Christmas Island and Southeast Asia (Indonesia, Thailand, West Malaysia, and East Malaysia) will be compared genetically and morphologically. Research Findings 2009‐2013 T. aurantiaca collected from 5 different sites and host plant species across Christmas Island are morphologically and genetically identical, based on cuticular morphology and mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S ribosomal RNA sequences (P. Gullan and L. Cook, personal communications, 2012). Initial morphological and genetic studies of T. aurantiaca collected from sites in Sarawak indicate that they are morphologically and genetically identical to T. aurantiaca on Christmas Island (P. Gullan, personal communication, 2012). Further study of Paratachardina pseudolobata was not pursued because, as pointed out above, it is not a source of useable honeydew for YCA on Christmas Island.

Key Research Outcome: The single population of T. aurantiaca on Christmas Island matches morphologically and genetically those populations examined so far within its native region in Southeast Asia. This establishes the identity of Tachardina aurantiaca on Christmas Island, making the successful establishment on Christmas Island of a biological control agent that attacks T. aurantiaca in its native range more likely. Further collections and analyses of T. aurantiaca in Eastern Malaysia (Sabah) and exploration in southern Thailand should establish if any morphological or genetic variation occurs across its native distribution in Southeast Asia.

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Project 2e. Scale population dynamics, especially the ecology of lac scale, Tachardina aurantiaca Pre‐release monitoring of T. aurantiaca and P. pseudolobata prior to any introduction and release of natural enemies so that the efficacy of natural enemies in controlling scale insect numbers and reducing yellow crazy ant densities can be determined. Research Findings 2009‐2013 The life cycle of Tachardina aurantiaca is relatively long, with one complete generation from F1 crawler settlement, through female maturation, to F2 crawler production and settlement completed between 88 ‐ 100 days, depending on host plant species (Table 5). Males emerge about 6 weeks after crawler settlement, consistent across host plant species (Table 5). On Christmas Island, there are overlapping generations such that all stages, including crawlers, female nymphs, and mature females can all be found together on twigs and along the midribs of leaves. This suggests that suitable life stages of T. aurantiaca susceptible to attack by Tachardiaephagus somervillei are available most of each year. Female size and offspring production vary considerably and is significantly correlated at the time of crawler release both in field and laboratory populations (R2 = 0.295, p < 0.001 in the laboratory and R2 = 0.207, p = 0.006 in the field) (Fig. 13). Large females can produce over 600 crawlers, indicating that conditions that affect female growth before crawler production could have a very strong effect on population growth. Conditions affecting grown are not well understood but could relate to settlement density of crawlers, position of crawler settlement on the host, host plant identity, and time of year. The role of males in reproduction is not clear, because it appears that some females are capable of producing offspring parthenogenetically (Ong and Neumann, unpublished data). The approximately 6‐ week development time of males suggests that females are mature at that time. Females apparently continue to live and grow well beyond the completion of development and offspring production occurs long after the female becomes mature (mating might not occur at all). Further experiments are under way to clarify the role of males in reproduction and the effect (if any) of parthenogenetic reproduction on life span and fecundity of T. aurantiaca.

Key Research Outcomes: The life cycle of Tachardina aurantiaca is relatively long for a scale insect but several overlapping generations occur in a single year. Female size is significantly correlated with fecundity, indicating that conditions that affect female size are likely to have a strong influence on population growth rates and density. The role of males in reproduction is not yet clear although it appears that some females can reproduce parthenogenetically. All of these attributes will influence host‐parasitoid dynamics on Christmas Island.

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Research Project 3. Strategic planning for biocontrol introduction Australia is well advanced in the regulation of import and release of exotic invertebrate biological control agents. Legislation stipulates import conditions, risk assessment, host‐ specificity test lists, release protocols, and pre‐release studies prior to approval of biological control introductions. Consultation with regulators early in the project is essential in the planning, conduct, and evaluation of research. Furthermore, early identification and agreement with co‐operators is essential for completion of research components key to proof of concept of indirect biological control of the YCA. Consultation outlined below will put project planning within the regulatory frameworks for biological control introductions in Australia and determine key co‐operators necessary to advance the project. Project 3a. Planning research in step with legislative and regulatory requirements for biological control introductions Smooth preparation and transparency in planning for introduction of biological control agents. Coordination of research findings in step with regulations (i.e. DAFF, DSEWPaC). Written report anticipating the regulatory steps and hurdles for advancement of the project. Research Findings 2009‐2013 The importation and release of an exotic biological control agent (BCA) on Christmas Island, an external territory of Australia, is regulated under Australian Government legislation by two independent, but parallel frameworks. The first framework falls under the Quarantine Act 1908, and subordinate legislation in the Quarantine (Christmas Island) Proclamation 2004. The process to seek approval to import and release an exotic biological control agent on Christmas Island is outlined by Biosecurity DAFF (Department of Agriculture, Fisheries and Forestry) in a document entitled Revised Biosecurity Guidelines for the introduction of exotic biological control agents for the control of weeds and plant pests (http://www.daff.gov.au/ba/reviews/biological_control_agents/protocol_for_biological_control_agents /guidelines‐introduction‐exotic‐bcas‐weed‐and‐plants Second, because the proposed action could have a significant impact on the environment, an Environmental Referral is required under legislation in the EPBC Act (1999). This framework is outlined by the Department of Sustainability, Environment, Water, Populations and Communities at http://www.environment.gov.au/epbc/assessments/index.html The criteria for evaluating importation and release of a BCA under the two frameworks differ somewhat. Under the Biosecurity DAFF framework, import risk analysis for a BCA differs from a standard commodity import analysis that assesses the probability of entry, establishment and spread. Because BCAs intended for release are deliberately introduced, risk analysis focuses on off‐target effects alone. Under the EPBC Act, DSEWPaC also has a linked approval process for the import and release of BCAs which uses the final risk analysis report produced by Biosecurity DAFF. This may be used by the DSEWPaC minister to include the BCA on the live import list. Under separate legislation under the EPBC Act, a proposed action is referred if it is likely to have a significant impact on a matter of national environmental significance (e.g. wetlands of international

24 significance, threatened species and ecological communities, migratory species). The process for submission and evaluation of an Environmental Referral is given at http://www.environment.gov.au/epbc/assessments/pubs/flow‐chart.pdf

Harmonization of these two frameworks is critical as we move through these regulatory processes. Key issues and Australian government contacts for progressing issues in each framework are given in Table 6. In discussions with Biosecurity DAFF, it was clear that they were unfamiliar with the Environmental Referral process (EACD) in DSEWPaC. It was equally clear that the Environmental Assessment and Compliance Division in DSEWPaC had not previously encountered Environmental Referrals that addressed importation and release of BCAs. A search of the 100 most recent environmental referrals indicated that 42% related to mineral or energy projects, 31% to infrastructure development, 20% to residential development, 5% to biodiversity management, and 2% to other. None of the Environmental Referrals since July 2000 on the referrals list page (http://www.environment.gov.au/cgi‐ bin/epbc/epbc_ap.pl?name=current_referrals&limit=999999&text_search=) addressed importation and release of a BCA. However, Biosecurity DAFF and the EACD DSEWPaC are now exchanging information on the CI project.

Key Research Outcomes: Two regulatory frameworks govern the importation and release of an exotic biological control agent on Christmas Island. To gain approval to import and release a biological control agent on Christmas Island, we will need to prepare and submit two sets of documents: an Approvals Package under the DAFF protocol and an Environmental Referral to EACD, DSEWPaC. The key government contacts involved in the two frameworks are described and currently consulting each other.

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Project 3b. Identification and agreement with co‐operators Identification of key co‐operators and agreements to advance the project. Cooperation will be necessary between Australian universities (e.g., project coordination, research staff mentoring and supervision); Australian federal agencies (e.g., assistance with regulatory issues in biocontrol introductions; taxonomic expertise on scale insects, ants, and parasitoids; Overseas universities (e.g., morphological and genetic studies of scale insects); Overseas government agencies (e.g., linked biocontrol program with foreign exploration and testing of natural enemies, biocontrol regulatory expertise ‐ USDA‐ARS, Invasive Plant Research Laboratory, FL; offshore facilities for safe host specificity testing – e.g., Malaysian Department of Agriculture) Research Findings 2009‐2013 We have established relationships with many researchers in Australia, India, Malaysia, Singapore and the United States, all of whom bring different expertise and capacity to this project (Table 7). Prof. Gullan and Dr. Cook provide taxonomic expertise on scale insects as well as morphological and genetic analyses for Tachardina aurantiaca on Christmas Island and in Southeast Asia. Dr. Hayat provides taxonomic expertise in the identification of encyrtid and aphelinid parasitoids associated with T. aurantiaca. A cooperative research agreement with Forestry Research Institute Malaysia provides benchspace and facilities for rearing T. aurantiaca and its natural enemies in Kepong, Selangor, Peninsular Malaysia. Ms Ong, a Masters student under supervision of Dr. Neumann, is conducting her research on the biology of T. aurantiaca and developing rearing techniques for it and one of its important natural enemies, Tachardiaephagus somervillei. Sarawak Forestry and the National University of Singapore provide laboratory space and field assistance. Dr. Pemberton, who has worked on the biological control of the invasive lobate lac scale in Florida, provides advice and was instrumental in establishing some of the cooperators listed in Table 7.

The complex permitting system in Malaysia has been negotiated successfully and permits to conduct research and collect specimens in West Malaysia, Sarawak, and Sabah have been granted.

An ad hoc advisory group of expert biocontrol practitioners has been assembled to review and advise on development of a host test list and a host‐specificity testing protocol (Table 8). Expertise in this group covers key aspects of in the development of biocontrol relevant to Christmas Island (e.g safety and risk assessment in biological control, host specificity of parasitoid natural enemies, biocontrol in natural areas, foreign exploration, scale insects as targets in biological control).

Key Research Outcomes: Collaborations have been established with researchers with expertise in the systematics, biology, and molecular genetics of scale insects, taxonomy of microhymenopteran parasitoids, and biological control of scale insects. Cooperative agreements have been established with FRIM and Sarawak Forestry that provide benchspace and technical assistance. Dr Neumann is serving as an advisor of a Masters student (Universiti Sain, Penang) who is conducting research on this project at FRIM. Research and collecting permits have been obtained from the States of Sarawak and Sabah, as well as a national collecting permit.

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Project 3c. Safety and sanitary protocols anticipating a pre‐release environmental assessment of introduction

An agreed list and protocol for host specificity testing of candidate bio‐control agents and protocols to eliminate and prevent the accidental introduction of hyperparasitoids to the island. Research Findings 2009‐2013 To focus host‐specificity testing for Tachardiaephagus somervillei (Hymenoptera: Encyrtidae), a candidate biological control agent for the invasive introduced yellow lac scale (Tachardina aurantiaca) (Hemiptera: Kerriidae), we used four approaches to assess the risk of importation and release of the candidate biological control agent T. somervillei to Christmas Island. Risk was defined as off‐target impacts, including those that could affect endemic species or threatened and endangered species. These risk evaluations were based on host records of Tachardiaephagus from the scientific literature, the diversity and attributes of other scale insects (superfamily Coccoidea) on the island, the occurrence of endemic hemipterans known from Christmas Island, and the host ranges of encyrtid parasitoids known to attack scale insects. All lines of evidence indicate that there is a very low likelihood that importation and release of T. somervillei would harm species of concern (i.e. those that occur on Christmas Island). We then describe a protocol for host‐specificity testing recommending that it be conducted within the area of origin (Malaysia) of Tachardina aurantiaca. Identification of an agreed host test list and protocol for host range testing of a candidate biological control agent are necessary requisites under the Biosecurity DAFF guidelines for the approval of importation of exotic biological control agents for the control of weeds and plant pests (see Project 3a). Lastly, in their native range, many primary parasitoids have their own insect natural enemies, including those known as hyperparasitoids (i.e. parasites of parasites). This is so for T. somervillei (Project 2a, Table 1). We describe standard rearing and sanitary techniques to ensure that the founding population from Malaysia is free of hyperparasitoids before importation to Christmas Island Known host range of Tachardiaephagus somervillei. We evaluated known host species records for all species of Tachardiaephagus using the Universal Chalcidoid Database (Noyes 2012) so that host range could be estimated and risk of release of T. somervillei assessed for species of concern on Christmas Island (see Sands and Van Driesche 2004). The Universal Chalcidoidea Databases (Noyes 2012) is the most comprehensive database for chalcidoid parasitoids, with over 120,000 host/associate records (including associations with food plants of the hosts) and > 140,000 distribution records of the parasitoids in the superfamily Chalcidoidea. It is very well developed, regularly updated and extremely well referenced. Nevertheless, large databases can contain errors affecting reliability, such as erroneous published host records and outdated parasitoid taxonomy (Kuhlmann et al. 2006). The most important source of error in this database is actual published erroneous records. Since all records are referenced, doubtful records can be investigated and if necessary, filtered out. Seven described species of Tachardiaephagus are distributed in Southeast and South Asia, and sub‐ Saharan Africa (Noyes 2012). Host records of Tachardiaephagus are restricted to species within three genera (Kerria, Tachardina, and Paratachardina) of lac insects (Kerriidae) (Project 2a, Table 3). Furthermore, all host species records for Tachardiaephagus somervillei are within two genera: Kerria, comprising the lac insects of commerce, and Tachardina. These records suggest that Tachardiaephagus somervillei is highly likely to have a narrow host range restricted to kerriid host species.

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Status of other scale insects on Christmas Island. We determined insect species on Christmas Island that could conceivably constitute non‐target species. Records for occurrence of scale insect species (Superfamily Coccoidea, the same superfamily to which Tachardina aurantiaca belongs) on the island were compiled from the literature and then supplemented by conducting >400 hours of structured search over two years for endemic scale insects (see Project 2b). A total of 24 species of scale insects in six families have been recorded on Christmas Island (see Project 2b, Table 4). All scale insects that could be considered as the most likely potential non‐target organisms are non‐natives on Christmas Island and none are known to be beneficial. No endemic scale insects were discovered. Phylogeny of endemic hemipterans. The closest endemic relatives of the yellow lac scale on Christmas Island should be considered to evaluate the risk of becoming non‐target hosts of Tachardiaephagus somervillei. We obtained a list of known endemic insect species on Christmas Island (James and Milly 2006) and narrowed consideration of taxa on this list to the closest endemic relatives of lac scales on Christmas Island in the Order Hemiptera, the same order to which the known target species Tachardina aurantiaca belongs. These species comprise one true bug, a cicada, a leafhopper, a spittlebug, and three planthoppers (Table 9). All of these endemic species occur in different suborders (either suborder Auchenorrhyncha or Heteroptera) than the yellow lac scale (suborder Sternorrhyncha).

Thus, any potential for non‐target impacts by T. somervillei on these endemic Hemiptera would require a host range that bridges this very substantial phylogenetic distance (see above and Fig. 2 in Cryan and Urban 2012), as well as distinctive morphologies, life‐histories and ecological attributes to its known host taxa in the Kerriidae. Host ranges of encyrtid parasitoids known to attack members of the hemipteran families with endemic species on Christmas Island. The Encyrtidae (Hymenoptera, Superfamily: Chalcidoidea), the family to which T. somervillei belongs, is one of the most important parasitic wasp (parasitoid) families for the biological control of harmful insects, including a variety of scale insects infesting woody plants (Noyes and Hayat 1994, Noyes 2012). The Encyrtidae currently comprises 460 genera and 3735 species in 2 subfamilies. The subfamily Encyrtinae includes 353 genera and 2920 species, while the Tetracneminae includes 107 genera and 815 species. Approximately half of all encyrtid species are associated with scale insects (Hemiptera: Coccoidea)(Noyes 2012). Encyrtids are generally endoparasitoids meaning that the parasitoid egg is laid directly inside the host’s body where the hatching larva completes development feeding on the host’s tissue, ultimately killing the host. Encyrtids mostly parasitize immature life stages (or, rarely, adults), but some species in one genus (Microterys) are egg predators (Noyes 2012). We used the Universal Chalcidoidea Database to analyze all records of encyrtid parasitoid associates of the hemipteran families represented by endemic species on Christmas Island. Next, all recorded primary hosts of encyrtid species associated with these taxa were determined. Consequently, any 28 encyrtid species that share (as hosts) both coccoid species (scale insects) and any family represented by endemic species on Christmas Island could be found. There are no known records in the Encyrtidae of any parasitoid species that attacks both scale insects (Coccoidea) and host species in hemipteran families that have endemic representatives on Christmas Island (Table 9). All known encyrtid primary parasitoids known to attack species in families which have endemic representatives on Christmas Island have primary hosts only within suborder Auchenorrhyncha. While species in these hemipteran families are attacked by many species of chalcidoid parasitoids, many fewer or no encyrtid species are known to use them as hosts, and not one encyrtid species is reported with a host range so extremely broad that it encompasses both scale insects and any of these hemipteran families. Host specificity testing protocol. Actual records for host range of Tachardiaephagus and known patterns of parasitism in the Encyrtidae indicate that it is highly improbable that T. somervillei would exhibit an extremely broad host range (one that would be unprecedented in any single species of Encyrtidae known to attack scale insects) and utilize any of the endemic hemipteran species on Christmas Island. Thus, when the host specificity testing of the biological control agent is initiated using the centrifugal phylogenetic approach, the outer boundaries of the sequential testing should be set initially at a significantly closer distance from the target organism than the taxa represented on Christmas Island by endemics. A ‘test species’ is an insect species that is tested as a potential non‐target insect. Since no non‐target scale insects were identified on Christmas Island, the list of test species will be determined using the centrifugal approach (Kuhlmann et al. 2006, Neumann et al. 2010). In this approach, species other than the known target host of the biological control agent are tested with the most closely related species (least phylogenetic distance, and with close similarities in biology and ecology) tested first then less similar species thereafter (“centrifugal principle” ‐ Wapshere 1974). A test species will be considered a ‘suitable host’ if parasitoid emergence is observed during any of the exposure tests. To be a suitable host, the parasitoid must (a) accept the test species when exposed, (b) must oviposit, (c) the eggs must hatch into larvae, (d) the larvae must complete development using the test species as the resource, (e) the larvae must pupate and, finally, (f) the emerging adult parasitoid must be able to exit (emerge from) the test species. If parasitoid emergence from a test species is observed, the test species will be considered a suitable host without assessing the viability, fecundity, sex ratio and other characteristics of the emerging parasitoid generation. A test species will be considered ‘susceptible’ if the parasitoid causes significant mortality due to probing, host feeding (when the parasitoid stabs the insect with its ovipositor and then feeds on the insect’s hemolymph), oviposition, or oviposition and larval development. Note that even if some mortality is observed due to the above, the test species will only be considered susceptible if the mortality resulting to the exposure to the parasitoid is significantly higher than in control groups (negative controls) of the test species where the test individuals are not exposed to the parasitoid. The ‘parasitoid’ in this case is T. somervillei (Hymenoptera: Encyrtidae), a widespread parasitoid native to Southeast Asia. T. somervillei is an important parasitoid of Tachardina aurantiaca (Project 2a, Table 1; Project 2c, Table 2). It is known to parasitize some scale insects in the family Kerriidae (lac scales) including the ‘target host’, Tachardina aurantiaca (Table 3). Selecting test species. Host specificity testing can be a long and difficult process, depending on how problematic it is to establish and manage colonies of the parasitoid, the target host, the host plant of 29 the target host, and the test species. Furthermore, resources and time are not unlimited and permitting issues to collect insects and run field experiments (when needed) in Malaysia can add to the time required to complete host testing. Therefore, a manageable number of species should be used (10 to 15, but fewer are acceptable if high specificity is found initially). The species used will be largely determined by availability, so determining the actual test species at species or genus level is not possible at this point. We can, however, predict to some degree the families of the test species that will be selected. We will focus on neococcid taxa including the Kerriidae, the family to which the target host belongs (see Fig. 14, also Gullan and Cook 2007, Ross et al. 2012). Considering the phylogenetic relationships of scale insect families (Fig. 14), we will aim to test one species in the Kerriidae, Paratachardina pseudolobata, which is also invasive on Christmas Island (see Project 2b, Table 4), and more than one species from the Coccidae. Species in the Diaspididae will also be considered as a less closely related group of scale insects. Early in the host testing process, an ‘out‐group’ (test species phylogenetically more distant) will also be used, most likely selected from the Pseudococcidae (Fig. 14). As mentioned above, availability will determine species selection to some degree but in some of the groups (e.g. Coccidae) there may be a number of species available. Species that can be reared in laboratory conditions would be preferred but carrying out tests in field conditions should also be acceptable as long as the tests can be well controlled. No‐choice tests. In no‐choice tests, only the non‐target test species will be provided to the parasitoid in the experimental replicates. This is the most stringent test method that places the parasitoid under high “oviposition pressure”. For each test species, trials will be replicated 10 times. Each replicate will consist of 1) one experimental cage with the test species (50 individuals in three different age groups/instars; 150 individuals in total) exposed to 10 female and 10 male parasitoids, 2) one positive control cage consisting of 50 mature female T. aurantiaca exposed to 5 female and 5 male parasitoids in order to confirm that the parasitoids used in the experiment are of good quality and capable of parasitism in the given exposure time, and 3) one negative control cage which will be similar to the experimental cage except the test species will not be exposed to parasitoids. The negative controls will be used to determine any effect of parasitoid exposure on insect mortality other than successful parasitization such as probing, host feeding and oviposition without successful parasitoid development. The cages will consist of fine mesh bags (sleeves) fixed on the branches of plants that serve as host plants for the scale insects. Parasitoids will be collected from a lab colony and introduced to the cages < 24 h after emergence. Exposure time to parasitoids will be determined during preliminary studies to determine the optimal time frame for mating, egg maturation and oviposition by the parasitoid. Evaluation: 1) If the positive control does not yield any parasitoid emergence, the replicate will not be evaluated (failed replicate). 2) If the positive control yields parasitoid emergence the replicate will be evaluated. The parasitization rate in the positive will be noted but will not be compared to parasitization rates in the test replicate. 3) Parasitoid emergence in the test replicate will be noted along with the approximate age and developmental stage of the individuals yielding parasitoid emergence. These individuals will not be used to compare mortality rates due to reasons other than successful parasitism. 4) Mortality of test species in test replicates and negative controls will be noted.

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Analysis: 1) If any test replicates yielded any number of parasitoid emergences, the test species will be considered a suitable host. The level of suitability of the test species will not be compared to the suitability of the target host at this stage of the project. 2) Mortality (except mortality due to successful parasitism) rates of test insects and insects in the negative control will be compared using 2‐sample t‐tests. If the mortality rate is significantly higher (α = 0.05), the test species will be considered susceptible and the reasons for mortality will be investigated. If a test species is susceptible (but not suitable), behavioural observations will be made to determine the cause of the effect of parasitoid exposure. The observational arena will be a ‘window box’ arena. This arena is constructed by constructing a 10 cm W x 10 cm L x 5 cm H box with one side being glass. The box is constructed so that it can be placed on the stem of a potted host plant on which the test insects feed. Female parasitoids are then liberated inside the box and the box closed. The potted plant then can be placed flat on its side and the box positioned under a dissecting microscope. Video footage is then captured using a high definition video camera with a microscope adaptor. The footage (4‐6 h per observation) can be analyzed later for the cause(s) of mortality (i.e. probing, host feeding, or oviposition without full parasitoid development). If oviposition is suspected, dissections will also be made to investigate parasitoid larval development. Other testing methods. We considered using choice tests (where the parasitoid is presented with the test species and the target host simultaneously) and sequential no‐choice tests (where the parasitoid is presented with the target host first then it is transferred on to the test species). However, in our case these tests may have limited value for reasons given below. A choice test would show whether a test species that is a suitable host in no‐choice tests would be consistently attacked when the target host is also available. A physiologically suitable host may not be preferred over the target host and the assumption could be made that parasitization would not occur (or at very low level) in field conditions. This test would be used in cases where the suitable host (found suitable in a no‐choice test) is of concern, e.g. a beneficial or endemic non‐target species that coexists with the target host. However, this situation does not arise on Christmas Island where all known scale insects are non‐native and none of them are beneficial. This test is further limited by the assumption that the test species and the target host occupy the same space hence providing the parasitoid with a choice. This would also not be the case on Christmas Island. A sequential no‐choice test would further show the unsuitability of a test species. The parasitoid may be ‘motivated’ to attack the test species even if it did not attack it in a simple no‐choice test if the parasitoid is first allowed to initiate oviposition behaviour and to gain experience. This test may be even more stringent than the simple no‐choice test and one would consider using it in the case of a test species that is of great concern to further show the unsuitability of the test species. This would again require a beneficial or endemic non‐target species neither of which exists on Christmas Island. Furthermore, similar to the choice test, this would assume either the coexistence or very close proximity of the test species and the target host. Options for the location for host‐specificity testing. There are three options for the location where host specificity testing could be conducted: 1) in quarantine containment at the release location (Christmas Island); 2) in quarantine containment on mainland Australia; and 3) in the native geographical range of the biological control agent where no containment would be necessary.

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Option 1. Testing in containment at the release location (Christmas Island). This option is the least attractive and involves increased risks and expenses. There is no quarantine containment facility on Christmas Island, a remote oceanic island. The expense of constructing such a facility to Quarantine Approved Premises Criteria (Quarantine Insectary Level 2) for host‐specificity testing of a single agent would be prohibitive. Even if there were such a facility, in case of escape, the biological control agent would find an environment very similar to its native range with its natural host T. aurantiaca in abundance. The benefits of this option would include easy access to the natural host for the parasitoid colony and no further travel if and when a release permit is obtained. Option 2. Testing in containment on mainland Australia. While this option is more attractive than Option 1 above, it is still somewhat risky, and labour and cost intensive. It brings few benefits. The biological control agent (parasitoid) would have to be brought into containment along with its natural host (Tachardina aurantiaca). This would pose the risk of not only a parasitoid escaping but also a potentially invasive, host‐generalist scale insect (on Christmas Island, for example, at least 15 horticultural species are attacked by T. aurantiaca, including three species of Citrus, Macadamia, Guava, Pomegranate, Chili, Eggplant, Star fruit, and Soursop)(R.W. Pemberton and D.J. O’Dowd, unpublished results). Using a containment facility in the temperate region of Australia could decrease these risks. In case of quality control issues of the biological control agent due to ‘lab selection,’ additional travel, collection, and importation of the biological control agent would be necessary, further increasing costs. Natural host colony loss (which can easily happen due to overexposure to the parasitoid agent, fungal infections, etc.) would necessitate multiple importations of the natural host, which would once again significantly increase costs and risk. Option 3. Testing in the native geographical range of the biological control agent with no containment necessary. In our case, this option is the most attractive, most cost‐effective, and least risky. There are many benefits to study biological control agents and carry out host specificity studies in the native geographical range of the agent. In our case, both the parasitoid and its natural host is readily available from the wild in Malaysia which ensures good quality parasitoid and host cultures. The risk factor would be zero as there would be no importation of any organisms. Although obviously desirable, such studies in the native range are not common, in part because of the lack of local research facilities, lack of skilled and reliable cooperators, or sufficient time in what are sometimes difficult locations (Van Driesche et al. 2008). However, scientific work is cost‐effective in Malaysia compared to Options 1 and 2 and our network of collaborators and cooperators is well established (Project 3b, Table 7). Options 1 (test in containment on Christmas Island) and 2 (test in containment on the Australian mainland) offer no benefits over this option. We therefore suggest that host specificity testing is carried out in the native range of the biological control agent. This option carries the least risk and is consistent with Recommendation 2 in a review of biosecurity risks in biological control commissioned by the Australian Government (Ferrar et al. 2004) that biological control practitioners undertake host specificity testing in the native region before any importation. Protocol for insuring that hyperparasitoids associated with T. somervillei are not imported to Christmas Island. The unintended importation of a hyperparasite of T. somervillei is inimical to the successful suppression of YCA supercolonies, because it could compromise the capacity of Tachardiaephagus to build up population densities sufficient to control Tachardina. For this reason, great care will be taken to implement standard agent rearing and sanitary techniques to ensure that the founding population of Tachardiaephagus from Malaysia is free of hyperparasitoids and pathogens.

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Free‐living adults are the safest to import because this would ensure that hyperparasitoids would not be co‐introduced. Tachardiaephagus somervillei is frequently attacked by Promuscidea unfasciativentris (Aphelinidae) (see Project 2a; Table 1). This hyperparasitoid was abundant near Kuching, Sarawak and present in Selangor, West Malaysia, although rare. The impact of hyperparasitism is not known; nevertheless, the exclusion of any hyperparasitoids from captive colonies in Malaysia is critical before adult T. somervillei are imported to Christmas Island as the founding population. The exclusion of hyperparasitoids from captive populations at locations where the hyperparasitoids are native can be difficult in the long run unless care is taken. Basic containment is usually sufficient but standard practices to keep hyperparasitoids out of captive populations must be followed. These include:

 Field collected, parasitized scale insects will never be exposed to laboratory scale insects directly; twigs with parasitized scale insects will be placed into emergence cages; emerging parasitoids will be examined individually before being removed from emergence cages.  If hyperparasitoids (or any organism other than T. somervillei) are found in the emergence cages, they will be destroyed.  Emergence cages will be kept in a dedicated room (the emergence room) in a separate building from that where the dedicated room with captive scale/parasitoid populations (the rearing facility) is located  T. somervillei will be moved to the rearing facility and placed on host plants with suitable scale insect hosts in a mesh bag; the mesh bag ensures that parasitoids remain on the plant and the scale insects are protected.  Mesh bags will not be removed (only shortly for monitoring purposes) from the plants until parasitoid emergence.  When parasitoid emergence is expected, host plants with parasitized scales in their mesh bags will be moved to the emergence room.  Personnel conducting field collections or any other field activity will not be allowed in the rearing facility on the same day. It is critical that the captive population is monitored continuously for hyperparasitoids even if best practices are followed closely. In addition to individual examinations of parasitoids, yellow sticky cards will be used in both the emergence room and the rearing facility to monitor (and also trap) any hyperparasitoids or other insects. Controlled exposures inside mesh bags will protect T. somervillei. The elimination of hyperparasitoids before importation to Christmas Island will involve the following steps. First, the sanitary practices followed during rearing (see above) will ensure a "clean" captive population. Second, emerged, adult parasitoids will be placed in airtight, glass vials. All individual parasitoids will be inspected during the packing and hand‐carried from Kuala Lumpur to Christmas Island Airport. All individuals will be examined again at the point of entry on Christmas Island while still in the glass vials (training of quarantine personnel on Christmas Island will be provided in advance); if in any doubt, the vial containing the questionable organism(s) will be destroyed. Third, an individual examination will be conducted in the rearing facility on Christmas Island before the parasitoids are removed from the glass vials.

In practice, the above procedures will ensure that hyperparasitoids are not transported to Christmas Island from Malaysia.

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Initial studies suggest that pathogens do not play a significant role in T. somervillei populations. In Malaysia, not a single T. somervillei, either field collected or lab reared, showed any signs of infection by entomopathogenic fungi or entomopathogenic nematodes.

Key Research Outcomes: an agreed, peer‐reviewed host list and host specificity protocol for evaluating risk of introduction of T. somervillei to Christmas Island to control the invasive yellow lac scale, Tachardina aurantiaca, with a recommendation that host specificity testing be conducted in the area of origin of Tachardina aurantiaca. A protocol for ensuring that hyperparasitoids are not accidentally co‐ introduced with importation of T. somervillei to Christmas Island.

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Project 3d. Rearing, release and monitoring protocols An agreed protocol for rearing and release of biological control agents, and pre‐ and post‐ release monitoring for their efficacy in controlling target scale insects. Research Findings 2009‐2013 Protocol for mass rearing of Tachardiaephagus in Malaysia (FRIM) and import this population into a mass rearing facility on Christmas Island. The transfer of the founder population from Malaysia to Christmas Island could be as simple as packaging freshly emerged adult T. somervillei inside a travel cooler to buffer them against extremes of temperature in transit. The parasitoids should survive well without access to food or water for 48 hours. There are direct flights between Kuala Lumpur and Christmas Island, but changes in scheduling may mean the parasitoids could be transported via Perth. This is the less desirable option because the probability of mortality is greater, and arrangements may have to be made with DAFF Biosecurity Australia to hold the agent in quarantine if there is a delay between flights. On Christmas Island, a production facility will have to be built to mass‐rear Tachardiaephagus for field release. As in Malaysia, the maintenance of captive parasitoid populations depends on the production and maintenance of optimal host life stages of Tachardina on suitable host plants. The best locally available host plant species for Tachardina are Inocarpus fagifer and Milletia pinnata. The major infrastructure item for this project will be a dedicated glasshouse/screenhouse (preferably closed, but solid‐roof screen house would probably be sufficient) for rearing of the biological control agent. Suitable host plants will be transferred to the glasshouse/screenhouse. If chemical control of pests and diseases on host plants is needed at the nursery, host plants must remain uninfested in the glasshouse/screenhouse until insecticidal residues are no longer active. Host plants can be inoculated with Tachardina within the same facility as long as plants in "waiting" and infested plants are separated with a barrier that prevents Tachardina crawler movement (e.g., a water moat system). Availability of a second, smaller glasshouse/screenhouse, to serve as a backup should the colony of Tachardiaephagus fail in the primary glasshouse, would be optimal, although not absolutely necessary. Tachardina and Tachardiaephagus may be difficult to mass‐rear due to the relatively long life cycle of Tachardina (Project 2e, Table 5 ‐ from crawler stage to reproductive female is 80‐100 days, but generations overlap) and the need for fresh host plants (that may or may not be reused) on a regular basis. Depending on the difficulty of rearing Tachardiaephagus, their availability for releases maybe limited at any given time. As mass‐rearing methods improve and production increases on Christmas Island, the goal will be to provide the biological control agent for releases in all areas as needed. The population of Tachardiaephagus will require careful monitoring and maintenance to minimize any selection of laboratory‐adapted insects that perform poorly under field conditions. It may be necessary to replenish the genetic diversity of the captive population through the subsequent introduction of more insects from the founder population in Malaysia, or, more likely, from the field on Christmas Island once the initial releases have been conducted. Population renewal will also counter the inherent susceptibility of microhymenoptera to the loss of population heterozygosity. Sex determination in microhymenoptera is usually haplodiploid – males are haploid, females diploid, and heterozygosity at a multi‐allelic sex‐determining locus is required for femaleness. Inbreeding can lead to a preponderance of homozygous diploids that will either be sterile males, or experience a very high rate of mortality.

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Field release of Tachardiaephagus. The goal in releasing biological control agents is to generate sufficient ‘propagule pressure’ (e.g., the size of each release, the frequency of releases, and the number and spatial arrangement of release sites) to enable their successful establishment. Increasing propagule pressure can enhance the likelihood of establishment by diminishing the role of chance (i.e., both demographic and environmental stochasticity), and potentially increase the rate of spread from release sites (Simberloff 2009). Initial timing, number of individuals released, and the frequency of releases of both agents will depend on (a) the capacity and sustainability of mass rearing, (b) knowledge of the biology of Tachardiaephagus, especially in relation to its host, Tachardina; and, (c) the attributes of release sites. Criteria for choosing suitable primary release sites will include: (a) positive evidence of host scale infestation; (b) relatively high percentage of host plants of Tachardina or soft scale in the overstorey and understorey; (c) occurrence of a high‐density YCA supercolony or at least the presence of YCA; and, (d) site not subjected to current pesticide exposure (contact or systemic) or residues that could compromise establishment of the agents. Some criteria (e.g., c and d) can be gleaned from the biennial Islandwide Survey (Green and O'Dowd 2009) and followed up by more detailed site assessments to determine (a) and (b). Each release at a site is likely to involve a specified number of adult insects; specific methodology of release will largely depend on knowledge currently being gathered about Tachardiaephagus in Malaysia and parasitoids of soft scale insects (e.g. Coccophagus) already present on Christmas Island. Fipronil, the intoxicant in Antoff®, the bait currently used to control YCA supercolonies on Christmas Island, is known to affect the longevity, fecundity, and behaviour of some parasitoids (e.g., fipronil used in vineyards to control ants can have acute toxic effects on Anagyrus sp. nr pseudococci and Coccidoxenoides perminutus, two microhymenopteran parasitoids of mealybugs – Mgocheki and Addison 2009). Thus, exposure of the biological control agents at release sites to baiting (especially aerial baiting where a fine dust is produced and a fraction of the bait is retained in the canopy) must be avoided. Coordination between field release and monitoring of biological control agents with National Parks staff involved in chemical control of YCA supercolonies will be critical during this phase of this project. Training on release methodology and criteria will be provided to National Park personnel. Some additions to the Island Wide Survey (e.g., determination of host tree species composition, inspection of understorey for Tachardina and soft scale insects) could facilitate selection of release sites. Interrogation of the survey database to identify the baiting history at waypoints will be an essential precursor to release of the biological control agents. The National Parks field crew will receive training in identifying and collecting scale insects and parasitoids. Monitor the establishment, spread, and impact of Tachardiaephagus and Coccophagus. The absence of effective, quantitative monitoring for the establishment, spread and impact of most introduced biological control agents has been the Achilles’ heel of many biological control programs (McEvoy 1996). Estimation of the success or failure of many past biological control programs has relied on subjective measures, often post hoc expert opinion alone (e.g., DeBach et al. 1971, Greathead 1989, Griffiths and Julien 1998). For biological control on Christmas Island, protocols to quantify the establishment, population status, spread, and impact of biological control agents are essential. Two approaches will be used. First, a field experiment will be conducted using a Before‐After‐Control‐ Impact design to determine the establishment and population dynamics of the agents, and the effect of

36 their release on host scale densities (counts per length of stem or per leaf) and parasitization rates, both in the canopy (random sampling of host plant material collected using a shotgun) and in the understorey (from saplings of known host trees), and abundance of YCA (using counts on tree trunks and on the forest floor) at release and control sites before and after release of biological control agents. Sites (each 2‐4 hectares) would be sampled 4 times before release of the agents and 4 times afterwards at two monthly intervals. Results will be analyzed as a one‐way repeated measures ANOVA, using release of the biological control agents as the main factor, and comparing response variables before and after release. In this design, the time x treatment interaction is the key term, with a significant difference in response variables after, but not before release. Thus, this experiment at the forest plot scale would establish both the outcome of the release and the mechanism(s) driving any change in YCA abundance. Second, at the much broader, island‐wide scale the outcome of agents releases on YCA supercolonies will be determined by comparing changes in YCA trunk traffic and ground activity (using card counts) at four‐month intervals at replicated release and control sites across the island. The number of control sites will be determined based on the release sites and area availability. Ideally, control sites should be distant enough from release sites so that the chances for biological control agent dispersal are low for a reasonable period of time. It will be necessary to determine how many of the selected release sites will be actually available for releases and the available areas for control sites where no other YCA management practices (i.e., application of toxic ant baits) will be applied. Spread of the biological control agents beyond release sites will be determined by placing potted ‘sentinel’ host plants, infested with Tachardina or coccoid scales, at set distances (probably at a logarithmic scale) from replicated release points, followed by their later collection to determine parasitization rates with distance from each release point. It may also be feasible to use the biennial Island Wide Survey to document spread of the biological control agent, at least onto understorey seedlings and saplings, at waypoints surrounding release sites.

Key Research Outcomes: Identification of the requirements for the establishment of a founder population of Tachardiaephagus in Malaysia free of pathogens and hyperparasites, importation of this population under permit to Christmas Island, establishment and expansion of this population in a mass‐ rearing facility on Christmas Island, an agreed protocol for rearing and release of biological control agents, and pre‐ and post‐release monitoring for their efficacy in controlling target scale insects and reducing densities and impacts of the invasive yellow crazy ant Anoplolepis gracilipes.

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Table 1. Natural enemy assemblages of the yellow lac scale Tachardina aurantiaca on Christmas Island and in Malaysia. + = present, ‐‐ = absent. For associates of T. aurantiaca, primary parasitoids oviposit on or in a host and develop within, ultimately killing the host. Hyperparasitoids seek out hosts with primary parasites, oviposit, and develop within the primary parasitoid. Predators feed externally and consume multiple scales.

Species (Family) Association with T. Christmas Island Malaysia aurantiaca

Tachardiaephagus somervillei primary parasitoid ‐‐ + Mahdihassan (Encyrtidae)

T. sarawakensis Hayat et al. primary parasitoid ‐‐ + (Encyrtidae)

Coccophagus euxanthodes Hayat primary parasitoid ‐‐ + et al. (Aphelinidae)

C. tschirchii Mahdihassan primary parasitoid ‐‐ + (Aphelinidae)

Coccophagus sp. (Aphelinidae)1 primary parasitoid2 ‐‐ +

Promuscidea unfasciativentris hyperparasitoid ‐‐ + Girault (Aphelinidae)

Aprostocetus (syn. Tetrastichus) hyperparasitoid3 ‐‐ + purpureus Cameron (Eulophidae)1

Marietta leopardina Motschulsky primary parasitoid4 + + (Aphelinidae)

Eublemma sp. (Noctuidae) predator + +

?Holcocera sp. (Blastobasidae) predator + +

1Tentative identification; 2Attack male T. aurantiaca only; 3primary parasitoid of many Coccidae, Diaspididae, Kerriidae, , and Pseudococcidae but known as a hyperparasitoid of C. tschirchii and Tachardiaephagus sp.; 4On Christmas Island and in Malaysia, Marietta leopardina is known only to attack male T. aurantiaca. It has never been observed emerging from female T. aurantiaca. In Southeast Asia, it is also a hyperparasitoid of primary parasitoids of a variety of scale insects.

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Table 2. Parasitization rates on mature females of Tachardina aurantiaca in the native range (Southeast Asia) and in the introduced range (Christmas Island). Parasitization rates were calculated as the proportion of mature female scale insects with one or more visible parasitoid emergence hole, either in isolated aggregates (N = 5) at sites in Southeast Asia or pooled within sites on Christmas Island (N is in brackets). This gives rates of parasitization at each site, but not the identity of the parasitoids. However, only T. somervillei was collected at sites in Kuching and Singapore. All other locations in Southeast Asia had a parasitoid assemblage of more than one species. For Christmas Island, number in parentheses after site name indicates the total number of mature females examined.

Rate of Parasitization Location (%, mean ± SE)

Native range (Southeast Asia)

Klang (Selangor, West Malaysia) 38 ± 17

Taman Ehsan (Selangor, West Malaysia) 46 ± 21

Singapore (National University Singapore campus) 73 ± 12

Kampung Istana, Kuching (Sarawak) 42 ± 23

Kampung Boyan, Kuching (Sarawak) 81 ± 6

Sandakan (Sabah) 76 ± 8

Sepilok (Sabah) 29 ± 13

Introduced Range (Christmas Island)

The Dales (Hugh’s – Sydney’s) (4000) 0 ± 0

Martin Point to CINP Boundary (1500) 0 ± 0

Dolly Beach Track (1000) 0 ± 0

North West Point Track (1500) 0 ± 0

Circuit Road (2000) 0 ± 0

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Table 3. Records of known host families and genera for the primary parasitoid Tachardiaephagus (Encyrtidae). Taxonomy follows Prinsloo (1977). The biological control agent under investigation, Tachardiaephagus somervillei, is in bold. As a genus, Tachardiaephagus has an extremely broad geographic range. With the exception of one probably erroneous host record in Africa (Prinsloo 1983), all Tachardiaephagus species appear to be family specialists and restricted to the Kerriidae. For host genera, number of species recorded as hosts is in parentheses. Based on Noyes (2012, Universal Chalcidoidea Database, http://www.nhm.ac.uk/research‐ curation/research/projects/chalcidoids/database/), except for records for T. somervillei and T. sarawakensis (Hayat et al. 2010; R.W. Pemberton, pers. comm.).

Parasitoid species Distribution Recorded hosts (all Kerriidae)

Tachardiaephagus India, Malaysia, Thailand Kerria spp. (4)1 somervillei Tachardina aurantiaca Tachardina sp.2

T. sarawakensis Sarawak (East Malaysia) Tachardina aurantiaca

T. tachardiae Brunei, China, India, Indonesia, Kerria spp. (8) Malaysia, Sri Lanka, Taiwan, Paratachardina lobata Vietnam, Azerbaijan

T. similis Afrotropical, South Africa Tachardina sp. (1)

T. absonus Afrotropical, South Africa Tachardina spp. (2)

T. communis Afrotropical, South Africa Tachardina spp. (5)

T. gracilis Afrotropical, South Africa Tachardina sp. (1)

1In Noyes (2012) both Kerria and Laccifer species are listed as hosts. However, Scalenet (Ben‐Dov et al. 2012) indicates that Laccifer is a synonym for Kerria, so we have synonymized these records with Kerria species. 2 Probably T. aurantiaca, since it is the only known Tachardina species in Asia.

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Table 4. Scale insects of Christmas Island. It is highly probable that all of these species, with broad host plant ranges and geographic distributions, are exotic to Christmas Island and introduced following human settlement. The target species, Tachardina aurantiaca, for biological control is in bold. Honeydew‐producing scale insects in bold occur commonly tended by YCA in supercolonies. Families are arranged in increasing phylogenetic distance from the Kerriidae based on Gullan and Cook (2007) and Ross et al. (2012). All scale insect taxa are 'neococcids' except for Icerya purchasi ('archeococcid'). Taxonomy and distributions from Ben‐Dov et al. (2012), http://www.sel.barc.usda. gov/scalenet/scalenet.htm).

Honeydew Family and Species1 Common Name Distribution Producer

Kerriidae (lac scales) Paratachardina pseudolobata False lobate lac scale Oriental, Nearctic, yes2 (Kondo & Gullan) Neotropical Tachardina aurantiaca (Cockerell) Yellow lac scale Oriental yes

Coccidae (soft scales) Ceroplastes ceriferus (Fabricius) Indian wax scale Cosmopolitan yes C. destructor Newstead White wax scale Afrotropical, Australasia, yes Oriental Coccus celatus De Lotto Green coffee scale Afrotropical, Australasia, yes Oriental C. hesperidium Linnaeus Brown soft scale Cosmopolitan yes Milviscutulus mangiferae (Green) Mango shield scale Cosmopolitan yes Parasaissetia nigra (Nietner) Nigra scale Cosmopolitan yes Pulvinaria urbicola Cockerell Urbicola soft scale Pantropical yes P. psidii Maskell3 Green shield scale Cosmopolitan yes Saissetia coffeae (Walker) Black olive scale Pantropical yes S. oleae (Olivier) Hemispherical scale Cosmopolitan yes

Diaspididae (armoured scales) Aspidiotus destructor (Signoret) Coconut scale Cosmopolitan no Hemiberlesia palmae (Cockerell) Tropical palm scale Cosmopolitan no Ischnaspis longirostris (Signoret) 3 Black thread scale Cosmopolitan no Lindingaspis sp. ‐‐ ‐‐ ‐‐ Pseudaulacaspis pentagona White peach scale Cosmopolitan no (Targioni Tozzetti) Unaspis citri (Comstock) White louse scale Cosmopolitan no

Cerococcidae (ornate pit scales) Cerococcus indicus (Maskell) Spiny brown coccid Cosmopolitan yes?

Pseudococcidae (mealybugs) Dysmicoccus finitimus Williams Asian coconut mealybug Australasia, Oriental yes

Ferrisia virgata (Cockerell) Striped mealybug Cosmopolitan yes Nipaecoccus viridis (Newstead) Spherical mealy bug Cosmopolitan yes Pseudococcus longispinus Long‐tailed mealy bug Cosmopolitan yes (Targioni Tozzetti)

Monophlebidae (giant scales) Icerya purchasi (Maskell) Cottony cushion scale Cosmopolitan yes 1Records from Campbell (1968), CSIRO (1999), O'Dowd et al. (2003), Bellis et al. (2004), Abbott (2004), Woods and Steiner (2012) and Neumann et al. (unpubl. results); 2Paratachardina pseudolobata produces honeydew but eject it instead of producing droplets that can be collected by ants (Howard et al. 2010) 3Tentative identifications 47

Table 5. Time (days ± SE) from crawler stage to emergence of either (a) adult males or (b) the production of the next generation of crawlers from adult females in Tachardina aurantiaca on seedlings of Acacia mangium x A. auriculiformis (n = 6), Milletia sp. (n = 7) and Inocarpus fagifer (n = 6) under laboratory conditions. All host plants were potted plants less than 1 m tall. Observations on hosts A. mangium x A. auriculiformis and Milletia sp. were conducted in at the Forest Research Institute of Malaysia in West Malaysia (Ong and Neumann, unpublished results). Observations on I. fagifer were conducted on Christmas Island.

Acacia mangium x A. Milletia sp. Inocarpus fagifer auriculiformis

(a) Days to male 40.1 ± 0.2 41.71 ± 0.13 42.5 ± 1.3 emergence

(b) Days to female 87.6 ± 0.2 98.3 ± 0.5 99.7 ± 1.2 crawler production

48

Table 6. Key Australian government contacts for progressing the importation and release of an exotic biological control agent on Christmas Island, Indian Ocean. Framework refers either to the DAFF protocol for biological control agents or the Environmental assessment procedure under the EPBC Act (EACD, DSEWPaC)

Framework Issue (Key URL) Section Key contact Addresses

DAFF Import risk analysis Grains & Forestry, Tara Dempsey [email protected] DAFF Assistant Manager, Processed (http://www.daff.gov.au/ba/reviews/biological [email protected] Products, Biologicals & Pacific Plant _control_agents/risk_analyses) Biosecurity, DAFF

Nomination of a target species for a biological Office of the Plant Susie Collins [email protected] control agent Protection Officer, Secretariat, Plant Health [email protected] (http://www.daff.gov.au/ba/reviews/biological Plant Health Committee, OCPPO _control_agents/protocol_for_biological_contr Committee [email protected] ol_agents/guidelines‐introduction‐exotic‐bcas‐ weed‐and‐plants)

Approvals package to import and release an exotic biological control agent (URL as for nomination) Amendment to live imports list Wildlife Trade Michelle van der Voort [email protected] (http://www.environment.gov.au/biodiversity/ Regulation, DSEWPaC Wildlife Trade Regulation Section, michelle.vandervoort@environment. wildlife‐trade/lists/import/index. html) DSEWPaC gov.au

EACD Environmental referral and assessment Environmental Felicity McLean [email protected] (http://www.environment.gov.au/epbc/assess Assessment and Assistant Secretary, Northwest [email protected] ments/index.html) Compliance Division, Section, EACD, DSEWPaC DSEWPaC

49

Table 7. Established collaborators and cooperators for the biological control project.

Location Collaborator Expertise

Australia ANU, Biological Sciences Prof. Penny Gullan Biology and phylogeny of scale insects

University of Queensland, Dr. Lyn Cook Phylogeny and genetics Biological Sciences of scale insects

India Aligarh Muslim University, Dr. Mohammed Hayat Microhymenopteran Aligarh, Uttar Pradesh taxonomy

Peninsular Malaysia Forestry Research Institute Dr. Laurence Kirton Entomology Malaysia (FRIM)

Universiti Sains Malaysia Ms Ong Su Ping Entomology (M.Sc student)

Sarawak Sarawak Forestry, Botanical Ms Lucy Chong Entomology Research Centre, Semenggoh Mr Het Bin Kaliang Entomology

Sabah Sabah Forestry Department, Dr. A. Chung Yaw Chyang Entomology Forest Research Centre, Dr. Chey Vun Khen Entomology Sepilok

Singapore National University of Dr. Rudolf Meier Entomology Singapore (NUS), Biological Dr. Hugh Tan Tiang Wah Entomology Sciences

United States

USDA‐ARS, Invasive Plant Dr. Robert Pemberton Biological control of Research Laboratory, Ft. insects and weeds in

Lauderdale FL natural areas, biosafety

50

Table 8. Expert ad hoc reviewers for the host test list and host‐specificity testing protocol (Project 3c). All of these colleagues have agreed to continue to advise us on this and other aspects of the project.

Reviewer Expertise

Australia Dr Don Sands OAM, CSIRO Biological control of scale insects Ecosystem Sciences, Brisbane QLD

New Zealand Dr Barbara Barratt, Principal Biosafety of biological control agents, risk Scientist, AgResearch Invermay, assessment, biocontrol in natural areas, Biocontrol, Biosecurity and developed the BIREA (Biocontrol Information Bioprocessing, Mosgiel, New Zealand Resource for EPA Applicants) for New Zealand

Switzerland Dr. Tim Haye, Commonwealth Host range assessment and impacts of Agricultural Bureaux International, parasitoids. Geneva

United States Dr. Robert Pemberton, USDA‐ARS Biological control of insects and weeds in natural Invasive Plant Research Laboratory, areas, biosafety, biocontrol of the lobate lac Ft. Lauderdale FL scale Dr. Matthew Purcell, Team Leader, Biological control of insects and weeds USDA‐ARS, Australian Biocontrol Laboratory, Brisbane QLD

51

Table 9. Endemic hemipteran species known from Christmas Island and primary parasitoids (superfamily Chalcidoidea: family Encyrtidae) associated with the families represented by the endemic species. The data were extracted from the Universal Chalcidoidea Database (Noyes 2012). The families Nogodonidae and Rhopalidae have no associated chalcidoid primary parasitoids and therefore endemic species in these families on Christmas Island can most likely be excluded from all further consideration. Cicadellidae and Delphacidae have the highest diversity of chalcidoid primary parasitoids but have magnitudes lower diversity of encyrtid primary parasitoids. These data suggest that the encyrtid primary parasitoids of families with endemic species on Christmas Island appear to not have host range overlap with taxa where the target lac scale is included and the host range separation is at the suborder level suggesting very distant phylogenetic separation. During the database analysis, only records with species‐level chalcidoid identification were used. N/A indicates not applicable

Endemic species Family No. chalcidoid No. encyrtid 1o Suborder/Family host associates parasitoid species range of encyrtids of family of family parasitizing family

Xestocephalus Cicadellidae 627 6 Auchenorrhyncha Izzardi1 (Cicadellidae) Oxypleura Cicadidae 35 0 N/A calypso Clovia eximia Cercopidae 71 4 Auchenorrhyncha (Cercopidae‐ Aphrophoridae) Ugyops aristella Delphacidae 248 5 Auchenorrhyncha (Delphacidae‐ Cicadellidae) Varcia Nogodinidae 0 N/A N/A flavicostalis Salona oceanica Leptocoris Rhopalidae 0 N/A N/A subrufescens2

1 Xestocephalus izzardi is also reported from Palau in the western Pacific Ocean (Linnavuori 1975). Its status as an endemic on Christmas Island is questionable. 2 Leptocoris subrufescens on Christmas Island has been classified to subspecies status (L. subrufescens subrufescens,). Another subspecies (L. s. flava) is described from Yap, western Pacific Ocean (Göllner‐ Scheiding 1980). More research is needed to resolve the taxonomic status of these two subspecies of L. subrufescens.

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100 90 2000

in

80 70 60 Tachardina

50 by 40 30 20

Contribution 10

% 0 70 80 90 100 110 120 130 Site Honeydew Index in 2000

Figure 1. The yellow lac scale Tachardina aurantiaca is estimated to contribute a large fraction of the honeydew economy at forest sites with YCA supercolonies. At each site the estimated range in % contribution to SHI by T. aurantiaca is given, based on per capita parity in honeydew production by T. aurantiaca and coccoid scale insects (blue circles, mean = 70%, range 46‐86%) or a 3 times greater per capita honeydew production by T. aurantiaca (red circles, mean 86.5%, range 72 – 95%).

53

80

70

60 Inocarpus 50 on

40

30 Tachardina

of

20 SHI 10

0 2002 2012

Figure 2. Estimated change in the Site Honeydew Index specifically for Tachardina aurantiaca on Inocarpus fagifer from 2000‐2012, at eight 0.25 ha sites in former YCA supercolonies. Numbers on the y‐axis are an index of Tachardina abundance and follow the methodology for calculation of the SHI, but applied only to Inocarpus.

54

3.6 Site 206 Site 318 3.4 Site 403 Site 582 3.2 Position

3.0

Trophic 2.8

2.6

2.4 1 10 100 1000 YCA abundance (no. ants 30 s‐1)

Figure 3. Stable isotope analyses (δ15N) of yellow crazy ants from four declining supercolonies showed that trophic position increased (i.e. YCA became more carnivorous) as population densities decreased over 16 months in 2010‐2011. In this figure, a trophic position > 3 indicates stronger carnivory while < 3 indicates increasing herbivory. The datapoints are connected in temporal order for each site, beginning at the right‐hand side.

55

A

50 45 B 1

‐ 40 s

30 35 ants 30 (no. 25

counts 20

15 trunk

YCA 10

5

0

Figure 4. Banding trees effectively excluded YCA from honeydew resources in the forest canopy (A), resulting in a precipitous decline and the virtual elimination of YCA traffic on tree boles 4 weeks after the tree bands were in place (B). Red symbols indicate control plot (unbanded trees), blue symbols indicate treatment plot (banded trees). Solid black line indicates when the bands were applied.

56

A

45 B 40 ) 1 ‐ s

35 30 1 ‐ 30 card

25 (n0.

20 counts

15 ground

10 YCA

5

0

Figure 5. Mean YCA abundance was estimated from replicate counts of the number of ants crossing one quadrant of a 20 cm x 20 cm card in 30 s (A). Exclusion of the yellow crazy ant from the forest canopy resulted in a significant and rapid decline in YCA abundance on the forest floor; YCA abundance fell and diverged markedly from the control plot two weeks after tree bands were placed (B). Red symbols indicate control plot (unbanded trees), blue symbols indicate treatment plot (banded trees). Solid black line indicates when the bands were applied.

57

0.08 A feeding

0.06

YCA

0.04 no.

Figure 6. Performance and foraging behaviour of yellow crazy ants depend on sugar supply. 0.02 capita

(A) Per capita recruitment to sugar indicated

Per that a smaller fraction of colony workers is 0 01234 needed to collect sugar with increasing sugar supply. Colony performance, as measured by (B) production of workers and males increased 600 P = 0.001 B with sugar availability whereas per capita 500 death rate decreased (C) with increasing sugar 400 availability. Per capita foraging tempo also increased with sugar supply (D).

individuals 300

200 new

No. 100

0 01234

0.5 C

dead 0.4

YCA 0.3

no. 0.2

capita 0.1 P = 0.025

Per 0 01234

0.025 P = 0.012 0.020 D exploring 0.015 object

0.010 no.YCA

novel 0.005 capita

0.000 Per 01234 log total sugar delivered (mg)

58

A

4 P < 0.001 spray

3 YCA

2 times

1 No.

0 012345

B 3.5 P = 0.077 3.0

2.5 megacephala

2.0 P.

1.5 dead

1.0 No. 0.5 012345 log total sugar delivered (mg)

Figure 7. Aggressive behaviours in the yellow crazy ant increased with sugar supply. YCA with access to more sugar sprayed P. megacephala with formic acid sooner more often (A), and killed more P. megacephala in 3:3 interaction trials (B).

59

Christmas Island

Figure 8. Christmas Island (red circle) is the only known area of introduction and invasion of the yellow lac scale Tachardina aurantiaca (Kerriidae). Yellow symbols indicate sites in Peninsular Malaysia (Penang Island, Klang, Selangor, Singapore) and Malaysian Borneo (Sarawak ‐ Kuching; Sabah ‐ Sandakan, Sepilok) where live aggregates of Tachardina were found.

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A B

Figure 9. Parasitism of the yellow lac scale Tachardina aurantiaca in Malaysia. A. Parasitoid emergence holes of Tachardiaephagus sp. (Encyrtidae) in tests of an aggregate of old adult females of T. aurantiaca on Milletia pinnata near Sandakan, Sabah, Malaysian Borneo. Yellow crazy ants (Anoplolepis gracilipes) tended T. aurantiaca at this site. B. Emergence holes of Coccophagus euxanthodes (Aphelinidae) in the test of a T. aurantiaca on Acacia auriculiformis at Klang, Selangor, Peninsular Malaysia. The smaller opening in the centre of the test is the anal pore through which honeydew is produced. Weaver ants (Oecophylla smaragdina) tended T. aurantiaca at Klang.

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1 mm

Figure 10. Tachardiaephagus somervillei, a primary parasite of the yellow lac scale Tachardina aurantiaca in Malaysia and Singapore (drawing from Narayanan 1962). In initial studies in 2011‐2013, T. somervillei attacked T. aurantiaca across Peninsular Malaysia and Malaysian Borneo, is the most abundant natural enemy of T. aurantiaca, exhibited superparasitism (i.e., where multiple progeny emergence from a single host individual), and heavily parasitized T. aurantiaca in the presence of tending ants, including the yellow crazy ant. T. somervillei can be reared under laboratory conditions (Ong and Neumann, unpublished results).

62

5

4 somervillei

T. 3 of

2 progeny

of

1 No.

0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Female T. aurantiaca size (mm)

Figure 11. The positive and significant relationship between female size of the yellow lac scale Tachardina aurantiaca and the number of progeny of the parasitoid Tachardiaephagus somervillei emerging from each host scale under field conditions in Malaysia (R2 = 0.471, N = 50 females, p < 0.01).

63

Figure 12. Search methodologies for detecting native or endemic scale insects and natural enemies of the yellow lac scale T. aurantiaca on Christmas Island. Northwest Point is enlarged in the upper left‐hand side of the figure.

64

700

600

500 female

/

400 crawlers

300 No. 200

100

0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Female size (mm)

Figure 13. Size‐dependent fecundity of female Tachardina aurantiaca under laboratory and field conditions, across a range of plant hosts. Female size was determined by measuring the test at the greatest horizontal diameter with callipers. The number of crawlers was determined by dissecting the females shortly before crawler release. There is a significant correlation between female size and number offspring produced (laboratory, open circles: R2 = 0.295, p < 0.001, n = 50; field solid circles: R2 = 0.207, p = 0.006, n = 50)(Ong and Neumann, unpublished results).

65

Pseudococcidae

Coccidae

Kerriidae Kermesidae Diaspididae

Gondwanan clade

BSE clade

Asterolecaniidae

Acanthococcid group

Dactylopiidae

Figure 14. Portion of a phylogram of the scale insects based on nucleotide sequencing of nuclear 18S rRNA for 72 species of scale insects and 10 outgroup taxa (see Gullan and Cook [2007] for details). Only the neococcoids (red) are shown here. For all families given here (except the Kermesidae), Bayesian posterior probabilities (numerical values above branches) are > 90%. For a host test list, we will select species from those families given in bold. The lac scales Kerriidae (bold and italics) is a sister family of the soft scales (Coccidae), more distant to the armoured scales (Diaspididae), and even further removed from the mealybugs (Pseudococcidae).

66

Christmas Island and Pulu Keeling National Parks Crazy Ant Scientific Advisory Panel (CASAP) Terms of Reference

1. Background

The introduced invasive yellow crazy ant (Anoplolepis gracilipes) is recognised in the Christmas Island Management Plan as the greatest threat to the Christmas Island ecosystem. In 2005, the yellow crazy ant was listed as a Key Threatening Process to Biodiversity in Christmas Island under the Environment Protection and Biodiversity Conservation Act 1999. The yellow crazy ant is also identified as a priority ant species in the Threat Abatement Plan that aims to reduce the impact of tramp ants on biodiversity in Australia and its Territories.

A ten-year Crazy Ant Management Strategy has been developed and includes scaling up the control effort and expediting a significant Research and Development program (including the development of biological control). Implementation of the strategy is critical to underpin long-term sustainable management of crazy ants on Christmas Island. In 2007, the Australian Government committed $4million over four years to implement the first stage of the Crazy Ant Management Strategy.

Likewise, yellow crazy ants are recognised in the Pulu Keeling National Park Management Plan as a potential threat to the biodiversity of Pulu Keeling National Park.

2. Purpose

The purpose of the Christmas Island Crazy Ant Scientific Advisory Panel (CASAP) is to provide scientific and technical advice to Parks Australia to inform pest ant management on Christmas Island and Pulu Keeling National Parks, with a particular focus on the implementation of the ten-year Crazy Ant Management Strategy for Christmas Island.

3. Terms of Reference

The CASAP will:  Provide scientific and technical advice on: - the impact of yellow crazy ants and their management on the ecology and biodiversity values of Christmas Island - management options for yellow crazy ants and other pest ants on Christmas and Pulu Keeling Islands -research and monitoring supporting pest ant management - integrating pest ant management with broader conservation management on Christmas Island and Pulu Keeling National Parks  Review the annual operational plans for yellow crazy ant management on Christmas Island and Pulu Keeling National Parks;  Review progress with yellow crazy ant research and monitoring on Christmas Island; and  Advise on matters relating to the National Tramp Ant Committee.

4. Membership

Dr Alan Andersen (Chair) CSIRO

Christmas Island Crazy Ant Scientific Advisory Panel (CASAP) Terms of Reference – 12/1/2009 Dr Dennis O’Dowd Monash University Dr Peter Green Latrobe University Mr Peter Davis Agriculture Western Australia Dr Kirsti Abbott Monash University Dr Hal Cogger Australian Museum Dr Ben Hoffmann CSIRO

Other members may be recommended by CASAP or the Christmas Island and Pulu Keeling National Parks Manager.

5. Observers

The following Parks Australia officers will participate in meetings as observers: The Director, Policy & Services; Manager, Christmas Island and Pulu Keeling National Parks; Natural Resources Manager, Christmas Island National Park; Yellow Crazy Ant Project Officer, Christmas Island National Park.

7. Secretariat

Parks Australia North is the Secretariat.

8. Meetings

Meetings are to be held twice a year, or more frequently as required.

9. Reporting

The CASAP provides advice to Parks Australia North through the Director, Policy & Services position.

The Director, Policy & Services acts as a conduit to senior management in Parks Australia and ensures that the CASAP’s views are appropriately considered in decision making and by island-based staff in their on-ground management of pest ants.

10. Relationship with the National Tramp Ant Committee (NTAC)

The relationship with the NTAC is managed through the Director, Policy & Services who liaises with the nominated DEWHA representative from that committee.

The Director is responsible for delivering a report from the NTAC at CASAP meetings and in return providing reports to the DEWHA nominated representative for presentation at the NTAC meetings.

Members of the NTAC that are also members of CASAP may provide additional support to this role.

Christmas Island Crazy Ant Scientific Advisory Panel (CASAP) Terms of Reference – 12/1/2009

Minutes from 12th December 2012 (CI time 10:00 am to 1.00 pm)

Venue: Dial in conference call – 10:00 am to 1.00 pm

Present: CASAP: Alan Andersen (AA, Chair) Kirsti Abbott (KA) Hal Cogger (HC) Peter Davis (PD) Peter Green (PG) Ben Hoffmann (BH) Dennis O’Dowd (DOD)

Observers: Mike Misso (MM: CINP/PKNP Manager) Samantha Flakus (SF: Natural Resource Manager) Dion Maple (DM: Invasive Species Project Officer) Ismail MacRae (IM: PKNP Chief Ranger) Tanya Detto (TD: Field Program Coordinator & minutes) Gabor Neumann (GN: Biocontrol Post Doc) *Pete Smith (PS: Field Supervisor) *Caitlyn Pink (CP: Field Officer) *Nina Trikojus (NT: Field Officer) (*Observers invited via AA).

Guest: Gary Morton (GM: YCA Eradication Program Coordinator, Biosecurity Australia) Lori Lach (LL: CFOC Tramp Ant Program Evaluation Consultancy)

Apologies: Minutes: Judy West Dion Maple Sarah Wittman Tanya Detto

1

1: MINUTES / ACTIONS ARISING FROM PREVIOUS MEETING Item 1.1: Apologies and adoption of agenda Judy West and Sara Wittman send their apologies. MM highlighted the need to discuss the scientific merits of the biocontrol research and development outcomes during the meeting and document reasons and support/lack of support by CASAP for proceeding into an implementation phase.

Item 1.2: Correspondence DM has been corresponding with LL regarding a CFOC tramp ant program evaluation consultancy and with GM regarding the Christmas Island and Queensland YCA programs as well as Phil Lester about the endoparasite work done by Monica Gruber and its applicability to Christmas Island. AA noted that Phil Lester would be in Darwin and would take the opportunity to talk to him.

Item 1.3: Ratification of the December 2011 Minutes The previous minutes were accepted without change or comment.

Item 1.4: Update of actions arising from December 2011 Action Item 1.4.1: Follow up on Monica Gruber’s endoparasite work on CINP DM is continuing to talk to Phil Lester about his research and Phil was under the impression that YCA on Christmas Island do not undergo unexplained collapse as recorded in Tokelau. PG was surprised to hear this as he has sent Monica Gruber data from colonies on CI that have undergone natural declines. LL believes that Phil Lester is aware of natural declines in YCA on CI in relation to scale insect abundance, but that these declines may differ to those in Tokelau based on his studies of their mitochondrial DNA. DM will continue to liaise with Phil Lester to develop potential collaboration with this research group.

Action Item 1.4.1: DM to continue to liaise with Phil Lester regarding YCA population dynamics.

Action Item 1.4.2: DM to finalise IWS report and forward to CASAP DM committed to finalising the 2011 IWS report before the next CASAP meeting and sooner internally. He reiterated that the YCA and red crab portion of the report were completed this time last year and that it is just a compilation of the other species that remains to be done. SW finished compiling the ants collected during survey in the last month, and the results will now be included in the report. All members accepted the delay.

Action Item 1.4.2: DM to finalise IWS report and forward to CASAP.

Action Item 1.4.3: Conversations to continue between researchers and managers regarding a project modelling the drivers of supercolony formation. DOD would like to see someone like PG more heavily involved in the projects given his extensive experience, and a greater combination of modelling and more empirical data. PG met with the students at the Ecological Society of Australia conference and is not overly happy with the way the

2

process has unfolded. The idea of modelling YCA spread is something he’s been talking about for a long time with DOD and KA and he is disappointed that the projects were developed in parallel but it hasn’t been suggested that they meet in the middle. He believes that while there is good data from the IWS showing where the supercolonies have and haven’t been, other empirical biological data is quite limited and they do not plan to conduct any field work. He believes that the power of mixing IWS data modelling with empirical studies can’t be underestimated. KA supports PG and is a little sceptical about how good the projects will be in filling in gaps in knowledge. She went on to state that combining empirical data collection with modelling would be ideal, particularly if someone like PG or herself could be involved as a co-supervisor. PG reiterated that some of the issues were well worth exploring, such as reducing the number of IWS waypoints while maintaining the necessary data, but that these aren’t PhD projects in themselves. HC added that the proposals were conceptually interesting but haven’t really considered the issue of uncertainty and need to test the correlation of factors before using habitat suitability maps. AA suggested that what was required was for PG, DOD and KA to meet with the students and supervisors for initial conversations and ongoing collaboration, and possibly do more field work. MM and DM agreed and made the point that the projects are still very much in the formative stage which provided opportunities to refine them. PG emphasised that the refining of the study could lead to some good outcomes and saw it as positive.

Action Item 1.4.3: PG, DOD, KA to continue discussions with PhD students and supervisors.

Action Item 1.4.4: DM to contact PD if any live Fipronil-resistant are found. No Fipronil-resistant beetles have been found alive since the bait was refrigerated. DM will inform PG if any beetles are found alive. Action completed.

Action Item 2.2.1. DOD and MM to produce referral under the EPBC Act and nomination to import/release the biocontrol agent and Action Item 6.1.1. LaTrobe and CINP to further discuss processes for seeking approvals to import/release bio-control agents PG stated that the purpose of the referral is to get clarity on the regulatory framework covering CI, which sits outside the normal mainland framework and confirmed that it is 99.9% complete and will send it to MM to review. DOD stated that it was MM’s idea to produce the referral in order to trigger department assessment processes and identify what regulatory framework CI falls under. MM added that it is important to prove that there are no significant threats to threatened species and Ramsar wetlands and show that CINP is required to abide by the same system and rules. DOD, DM, GN, SF and MM met on Christmas Island in September to discuss the regulatory steps required if the biocontrol program moves into the implementation phase. GN produced a protocol to test for host specificity. DOD is exploring the Biological Control Act passed in 1984 which allows the Minister for DAFF to declare that external territories are part of Australia so CI can fall under the regulatory framework of the mainland. Need someone who is able to talk to DAFF and Biosecurity and MM thinks that the referral is a good way to achieve that as it puts the onus back on the department to determine/identify what other approval processes may be needed.

Action Item 1.4.4: PG to forward completed referral to MM for review.

Action Item 2.4.1. DOD, PG, GN to produce a report on the status of the biocontrol program and include suggestions for a survey to monitor its success Action completed, Executive Summary finalised and disseminated to CASAP. Discussion undertaken and in-principle decision made in Items 2.3 and 2.4.

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Action Item 2.7.1: AA to draft dot points of a rebuttal to the CIP paper and circulate to CASAP and JW The dot points were sent to JW. MM stated that it was important to have the information for future use if needed for other documents. Action completed.

2: CHRISTMAS ISLAND Item 2.1: Bio-control project updates (YCA/Scale) DOD stated that this item is subsumed in upcoming agenda items. MM took the opportunity to thank SW for her work on behalf of CINP over the last 3 years. DOD offered to draft a thankyou letter on behalf of CASAP, AA will send it on.

Action Item 2.1.1: DOD to prepare thankyou letter for SW, AA to forward it

Item 2.2: Bio-control project updates (Agent) This item was discussed as part of Items 2.3 and 2.4

Item 2.3: Bio-control Research and Development Executive Summary DM sent the Executive Summary to CASAP on the 23rd November for input on a preliminary decision. AA asked DM and MM for clarification on its progress. MM stated that he, JW, SF and DM had spoken yesterday and the Executive Summary covered all the necessary points. One of the things requested from DOD and PG was a risk assessment that included the risks of doing nothing (i.e. not implementing bio-control). MM would like CASAP to discuss whether the introduction is likely to have an impact on threatened/non-target species, clear views on the feasibility of success and the scientific merit of the research. AA asked DOD and PG to excuse themselves from the subsequent discussion and MM stated that this was previously requested by DOD and PG to avoid any previously acknowledged potential conflict of interest. They were to call back in 10 minutes and to e-mail GN to call in at that point.

Item 2.4: Bio-control Implementation Phase Decision/Discussion KA indicated that she was impressed with the research and development phase of the bio-control program and believed that the scientific merits of the program were high. She had no problems with the way the research had been executed and believed that SW and GN had demonstrated the proof of concept well. The work of SW and GN combined says that success is likely overall. Based on her own work within the agricultural field, KA didn’t seem to think that the risks to non-target species would be an issue, especially considering the number of invasive species on island. She also thinks that it is highly feasible, based on agricultural experience, and believes they have covered everything although the complexity of the rainforest and hazards of access means it will be difficult to check the whole island. KA registered absolute positivity and wholeheartedly endorses it. HC is concerned with one statement and would change “negligible” to “low” in the sentence “assuming any prospective BCA would be a family specialist... negligible” as it is rather dismissive of the possibility of impacts on non-target species. In general terms he found it very convincing and seems to have covered all the issues and he gives it a general tick. PD stated that the research was good and compared to other options is a relatively positive option with likely much lower side effects. With regards to the risk he made the point that it is important to

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assess the risk against the current bio-security measures and thought that the risks associated with introducing a bio-control agent was low, particularly given the poor bio-security controls currently in place where things could easily be introduced (i.e. parasites could be introduced on the next introduction of scale anyway). He questioned why the regulatory and referral processes had progressed without CASAP advice to proceed. MM clarified that, due to time constraints, it was decided that these processes would run in parallel so that they were ready for submission once the advice of CASAP and approval from the DNP were received. He explained that while normally it would be a linear process, we were trying to expedite the process by doing several steps in parallel to keep the process moving. BH noted that he had already sent his comments directly to DM but reiterated that he was impressed with the outcomes of the research and endorses going forward with implementation. He also believed that the risks of not moving forward with bio-control was much higher for the ecosystem health than the risks of introducing the proposed agent. AA stated that there is pretty clear endorsement from the panel and asked if MM was happy with the advice. MM asked if it was possible to get a one page formal statement of advice (based on the minutes), similar to that regarding the CIP Borax trials and said that the issue PD raised in regards to the comparative bio-security risks would be good to include. Following these discussions, DOD, PG and GN re-entered the conversation and were informed that CASAP fully endorsed their work and with proceeding with implementation. PG thanked the members for reading and approving the continuation of the program and asked the panel members to treat the Executive Summary as confidential. He went on to ask what the panel members thought about the possibility of targeting the soft scales as well, which he says will give a greater degree of certainty to the program. AA asked PG for his thoughts on what should happen. PG explained that they were pretty sure that Tachardina is a major contributor of honeydew. Part of SW’s work was to make fine-scale measurements of per-capita rates of energy content of honeydew and looking at relative contributions of different scale species but she was unable to complete this. The report gives a best estimate of 70% contribution of honeydew by Tachardina but this is based on older data. PG indicated that we may not see a consistent depression of YCA across the island even if we manage to control Tachardina. This depression would be enhanced considerably if we went after soft scale as well. GN already found 2 parasitoid predators on-island so it wouldn’t involve additional host specificity testing or import processes. There was some discussion as to the effectiveness of these predators to date but it was highlighted that their dispersal capabilities may have limited them from spreading. However it has been demonstrated that they are successful when cultured and distributed to infested areas. There is an opportunity to get a better effect by including soft scales in this program. AA asked for an indication of the resourcing/time/personnel required. GN stated that he didn’t think any extra resources would be required to include other predators with the Tachardina control. AA asked whether the program would be ongoing due to the dispersal limitations and GN responded by saying that he doesn’t understand why the parasitoids are mostly around settled areas but has some suspicion that it may be due to fipronil application which is highly toxic to the parasitoids. He has asked Cesar consultants to provide him with data relating to the parasitoid families (there are 4 now as he found 2 more species) in the next few weeks. He found that the Pulvinaria parasitoids established everywhere he inoculated, except in areas where they wiped out the scale completely. He believes that it wouldn’t be necessary to introduce the parasitoids more than once at each site, but he can’t say with absolute certainty. DM noted that Pulvinaria parasitoids have been found in areas where they hadn’t been released (on the east coast of Egeria) which is proof that they can move on their own. PG reiterated that the figure of 70% presented in the Executive Summary is based on data from 2000 and focussed on the role of Inocarpus as a host for Tachardina. Recent work has found a stand-level decline of Inocarpus in many areas so the balance may already have shifted from Tachardina to soft scale, which is more reason to consider going after soft scales as well.

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KA stated that the decline in Inocarpus and the production of large quantities of honeydew by soft scale are both good reasons to go after them. Considering that no extra resources are required, she thinks going after soft scales is a really good idea. HC agrees and can’t see any disadvantages to going after soft scale. PD asked if there was any opportunity to coordinate the releases of the parasites with the baiting program and asked whether the continued decline of Inocarpus after baiting could be due to the removal of ants removing honeydew resulting in an outbreak of sooty mould. PD suggested that there might be a post-baiting explosion of Camponotus tending Tachardina after baiting as they found Camponotus at many sites where Inocarpus were gone. DOD mentioned the lousy documentation of the phenomenon and PG stated that next time he is on-island he will visit baited sites to study how quickly they may take over. DM stated that for interest’s sake there is a super dense colony of Camponotus on the Boulder Track. BH said that targeting soft scale is a great idea as it covers more options, removes uncertainty and gives you 2 for the price of 1. DOD went on to say that soft scale are not a new emerging issue, they were incorporated in the original proposal and the 10 year plan. They went after Tachardina because they could and the options of dealing with soft scale were limited because there were no agents on-island and the expertise at the time said to go after Tachardina. AA summarised by saying that everyone supported including soft scale since it is feasible and requires no extra resources and asked for comments from DM and MM. DM and MM both were supportive and DM added that Parks had tried raising Coccophagus but found it difficult without someone dedicated to the task but it was good practice. MM requested that AA include this discussion in the statement of endorsement.

Action Item 2.4.1: AA to draft a formal statement of endorsement to progress to the implementation phase of bio-control and circulate to CASAP for comment before providing to CINP.

Item 2.5: Aerial Baiting Campaign Report DM took the time to thank his team for their work, especially given the area of supercolonies baited. AA asked for clarification on the use of multiple chemicals per supercolony. DM explained that supercolonies that extended from the coast to the terraces were treated with fipronil 200m from water sources, and the 2 IGRs in areas closer to the water. AA clarified that he meant different areas within a supercolony were treated with different chemicals rather than the same areas getting multiple treatments. AA went on to ask whether the explanation for the strange results in supercolony 140 was based on real or theoretical knowledge of problems with the treatment. DM reported that there were issues with the flow of the bait and an error in the maps given to the pilots as well as mechanical problems with the flow recorder on the chopper that led him to believe the area may have received the wrong treatment, but there is no way to be sure. DOD is troubled by Figure 11 and the big increases in ant densities in IGR sites. DM stated that the densities weren’t that extreme pre-baiting but since the first rain the numbers have just exploded, also the crab migration is underway so the team put fencing around the colony to divert the crabs. He is not sure when, but plans to deal with some of these colonies fairly quickly. DOD asked how the 80% reductions in ant densities compared to the 2009 baiting. DM attributed the slower rate of decline to a far greater density of ants in the colonies this time around. Supercolony 184 in 2009 had similar densities to this year but it got a second treatment and this year we couldn’t retreat any supercolonies. He went on to say that while the declines aren’t as marked, they’re not too bad.

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DOD asked if he got a chance to look at the monopolisation of bait which he raised in the last meeting. DM responded that he simply ran out of time to design then implement research for this and other items due to the unexpected size of the supercolonies. BH asked whether the wind speed was measured as the flight paths are very impressive, either because it wasn’t windy or perhaps just showing the best ones? DM responded that the flight paths were fairly representative and the wind was fairly gusty but typical for that time of year. PD shared DOD’s concern about the effectiveness of the 2012 baiting campaign, given the success of previous aerial baiting campaigns being in the order of 99%. He queried whether the lower results might correspond to the use of 2009 bait and asked if the bait had been analysed for active ingredients. DM noted that the old bait was analysed prior to the campaign and while there were some mixed results on its quality, he highlighted that the old bait was generally mixed with new bait and for best results. It was also noted that some areas were baited solely with the old bait and still had effective control. PD reiterated that it was another hell of a good job. BH stated that the IGR treatments had been done again when the queens were in their pupal state (i.e. September) so he was not surprised that the impact wasn’t as good as in the past. KA added that un-winged queens peaked in their pupae state around October. KA would like figures 10 and 11 to include card counts (rather than percentage decline) since it’s difficult to determine what “densities” really mean. She asked whether supercolony definitions are still 37 ants? DM responded that initially the definition is 37 ants but when doing boundaries we use other criteria. He went on to say that he could provide revised graphs with ant counts and would replace the phrase “density” with “ant activity”. PG and HC had few questions about figures that were already answered. MM took the opportunity to thank DM and the rest of the team for the large effort conducting the aerial baiting over a short time frame, especially given the large increase in super-colony estimates since the 2011 IWS and the baiting.

Item 2.6: CINP Natural Resource Management Programs Update SF had nothing else to add to the report except to highlight the decline of flying fox detections by 39% in the drive survey. AA asked for clarification on the range data for the cat removal. SF stated that there was certainty in the number of cats removed by traps but not by baits. KA enjoyed reading about the other work being done but asked why the biocontrol work isn’t included in the table of research being conducted. SF stated that she based the information in the table on the current research permits that had been issued and the biocontrol work doesn’t require a permit. KA would like to see it included with the other work being done. HC asked how the level of bait-related robber crab mortality compared to previous years. DM responded that the study undertaken by CP found a total of 5 deaths in 12ha studied and that the feeling was that mortality was low across the island, probably due to the extremely dry conditions. PD commented on the large number of cats being removed, SF added that one of the good outcomes is an increase in tropicbird nest success and that we hope to continue the program if funding applications are successful. PG said that it was great to see the programs continuing and is very concerned about the flying foxes and asked if anyone understood the drivers of the decline. SF explained that a recent meeting with mammal experts and the decision making hub from NERP examined the potential drivers/threats and identified ways to confirm or rule out some of these threats. PG went on to say that the reptile program seems to be kicking some goals and is horrified by the number of robber crabs being killed on the roads. He asked if it was worth the Minister for Environment questioning the Minister for Immigration. He went on to say that he knows people on the island are doing everything possible to educate people, but the message doesn’t seem to be getting through. MM responded that he met with Minister Crean who was also concerned and that

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the head of DIAC was very supportive of the work of Parks and happy to comply with the information sent out. He thinks that the program is working but agrees that it’s an unacceptable level. A recent incident of someone from SERCO reported deliberately throwing robber crabs is being investigated and the person has been laid off until further notice. He thinks that the subcontractors are more of a problem than DIAC or SERCO staff, and that it seems that awareness is the key issue. BH and DOD both appreciated reading about the work. DOD suggested adding a paragraph on the YCA work to give a whole panoply of the work being done on CI. HC made the point, in reference to Tim Flannery’s paper, that these will be the first reptiles to go extinct since settlement of Australia.

Item 2.7: CINP Management Plan and Regional Recovery Plan Update MM explained that the Management Plan has been modified based on the 11 submissions received and is now subject to the approval processes of the Director of National Parks (DNP) and the Minister. With regards to content, it is pretty much there, subject to final review by the DNP. There is also not much to report on the Recovery Plan, as progress has slowed down, partly due to some concerns on-island.

3: PULU KEELING Item 3.1: PKNP Natural Resource Management Programs Update IM informed the panel that invasive species monitoring of Pulu Keeling continues whenever they get a chance to go to PK. There was a buff-banded rail translocation meeting on Monday which resolved that WA DEC needs to do a final check of the rat control program on Direction Island before a timeline for the translocation program can be established. The panel members expressed their support of the programs.

4: National Pest Ant Management Item 4.1: National Pest Ant Management Developments PD and AA suggested removing this item from future agendas as there is no national pest ant management program.

Item 4.2: YCA Control in Wet Tropics. Queensland Program Update GM informed the panel that there is no YCA program in the wet tropics at the moment as the program was cut shortly after the last meeting. He has been unable to do follow up aerial treatment. Despite the absence of staff he has been able to do some assessment of baited sites and the infestation hasn’t expanded from before the treatment. The YCA program has been moved to a different part of Biosecurity Queensland, who agreed to a one off final treatment of hand baiting with left over fipronil in the rainforest, and using bait stations in the sugarcane. Biosecurity Queensland’s position is that eradication is not feasible so they will focus on controlling them on people’s land rather than eradication. AA asked for the panel’s thoughts on the eradication not being feasible. GM stated that eradication is not feasible with the resources available. He has submitted an application to Biodiversity Fund to eradicate YCA from the Cairns area. Wet Tropics Management Authority has also put in a proposal for a Caring for our Country grant for surveillance, working with Conservation Volunteers and the Federal Government has made some initial sort of commitments in this area.

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PD asked about the assessment of IGR treatments and GM responded that he was able to do a quick pre-treatment surveillance but had no staff to do anything after that, although it is part of the Wet Tropics Management Authority proposal. The rest of the panel expressed their disappointment and BH asked whether the pest risk assessment has been completed. BH pushed hard to get the NT included as well but hasn’t seen the report and has only seen a press release criticising the report. GM hasn’t seen the report either but has received some data from George Anthony. GM went on to say that the sugar industry may get more involved, as the sugarcane is infested near the World Heritage Areas, and he hopes that pressure from the agriculture industry might affect things.

Item 4.3: Tramp Ant Consultancy LL informed the panel that she is collating all the results but at this point under the confidentiality requirement she is unable to say any more than is in the attachment. DOD stated that the threat abatement plan from 2 years ago was supposed to get a whole of government approach but since there was no funding framework, there seems to be no material result. PD asked what the eventual fate of the report would be and whether it would be made public. LL responded that she was unable to get a clear answer to the same questions, but believes that the whole report would be sent to all the programs involved. Her inclination would be to include as much detail as possible. PD asked when the report was due and LL responded, mid-January due to changes in staffing. HC asked whether there would be a real ranking process in terms of program effectiveness to identify the elements of a successful program that can be used in the future. LL replied that that would be the aim, recognising that a lot is context-dependent; it would be good to know what lessons have been learned and the future approach. KA asked to what extent the report is incorporating the recommendations of the threat abatement plan. LL said that is will be incorporated as much as possible and point out where recommendations should be followed that weren’t. KA added that with 76 recommendations is will be difficult to incorporate them all, but look at what worked. AA asked if any members wanted to discuss the lack of a national tramp ant program. DOD and BH had nothing more to add. PG asked whether the threat abatement plan influences the programs and asked whether this was part of the review. LL responded that it wasn’t but that were certainly drawing on it. PG advocates a national approach. PD reiterated that a lot of work goes into producing a threat abatement plan but then it is not really followed up, it seems to be an exercise in appearing to be doing something. He suggested that perhaps it can be used, with the cancelling of the Queensland program and the review, to ask questions of the national government. LL had no further questions for the full committee.

5: Arnhem Land Item 5.1: YCA Program in the Northern Territory Update BH informed the panel that he is in Nhulunbuy, ready to start treatment of the largest area (600ha) next year. He has been working with Animal Control Technologies to get the bait dry enough that it flows freely without further drying. The conditions are perfect; dry with no wind. He has also been in contact with a colleague from New Caledonia who will be coming to Darwin to help out with the baiting and learn the process. He went on to ask DM whether he had seen queens flying like last time, DM responded that he had seen them possibly around November. PD asked if there was any significance in the possible closure of the processing plant. BH responded that no one really knows but it would only be the refinery, not the mine, if they do close something. He suspects that if the mine continues running it will have to continue rehabilitation so

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they probably won’t be in a bad situation, whatever happens. He had tried to get them to increase the financial contributions but was unsuccessful but most of the Arnhem Land YCA money is federally funded anyway.

6: Other Business Item 6.1: Advice on Pre-treatment Scale Assessment Methods for the 2013 IWS DM explained that he would like the panel to consider the requirements for pre-biocontrol scale assessment and how this might be achieved, and if it is possible to incorporate scale assessments into next year’s IWS. GN responded that it can be quite complicated, especially if you want to assess all species. It’s much easier if just focussing on Tachardina. It’s possible to use a slingshot (time-consuming) or a shotgun (obvious safety issues) to collect scale from the canopy or collect crawlers that fall from the trees, but only when density is very high. It’s also possible to use some indirect methods like using tree types and ant tree traffic to examine scale species and density, but the IWS is already an enormous effort and he can’t see how you can assess the whole island for scale insects. PD reported using a fishing line to get a saw blade into the canopy to fell branches. PG said that even on his best day he could only slingshot 15 trees a day, a shotgun would be more efficient but he agrees with GN that it’s hard to do directly and not possible to use the IWS for pre- and post-monitoring. It was suggested that only certain sites be chosen and that PG and DOD put a lot of thought into the idea to develop a monitoring program before the IWS. HC asked if there was any advantage in identifying which waypoints would be suitable for upcoming survey. PG said that planning ahead for pre-release monitoring will be quite tricky. GN agreed that determining the waypoint locations will need to be done 5 or 6 days before the release to determine what life stage the scale insects are at, so the parasitoids can establish. AA went on to say that a very broad scale island-wide assessment would presumably be required in parallel with dedicated, targeted monitoring. GN suggested incorporating a search of the vegetation, especially Tachardina host plants, and note ant traffic on those trees at each waypoint, which should only add 5 minutes to the survey.

Action Item 6.1.1: PG and DOD to include any scale assessment needs of CINP into the biocontrol implementation scope of works.

7: NEXT MEETING Item 7.1: Date for next meeting DM suggested late April, just before the survey, as a potential date for the next meeting. AA asked the panel for the opinions on an on-island meeting given the last one was in 2008. DM said he would have to check the budget. MM said it would be good to value add to existing visits that may be coming up. DOD stated that he goes to the island fairly regularly but it would be good to have some face-to-face contact. BH agrees, but is unavailable for the next meeting. PG and PD both agreed. HC thinks it is fantastic but just came back from the island so can’t argue for it strongly. AA suggested meeting at the end of the year and HC agreed that there would be more information to discuss after the survey. The panel concluded by thanking everyone for their hard work and MM thanked the panel for their contributions given members are busy and have other jobs.

Action Item 7.1.1: DM to organise next CASAP meeting for April 2013 Action Item 7.1.2: DM and MM to look at possibility of on-island meeting at the end of 2013

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Action Item Table Item Who Description

1.4.1 DM DM to continue to liaise with Phil Lester regarding YCA population dynamics.

1.4.2 DM DM to finalise IWS report and forward to CASAP.

PG, DOD, 1.4.3 KA PG, DOD, KA to continue discussions with PhD students and supervisors.

1.4.4 PG PG to forward completed referral to MM for review.

2.1.1 DOD, AA DOD to prepare thankyou letter for SW, AA to forward it

AA to draft a formal statement of endorsement to progress to the implementation 2.4.1 AA phase of bio-control and circulate to CASAP for comment before providing to CINP.

PG and DOD to include any scale assessment needs of CINP into the biocontrol 6.1.1 PG, DOD implementation scope of works

7.1.1 DM DM to organise next CASAP meeting for April 2013

7.1.2 DM, MM DM and MM to look at possibility of on-island meeting at the end of 2013

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