Biology, host specificity, release and establishment of Macaria pallidata and Leuciris fimbriaria (Lepidoptera: Geometridae), biological control agents of the weed Mimosa pigra

Biological Control 55 (2010) 248–255

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Biology, host specificity, release and establishment of Macaria pallidata and Leuciris fimbriaria (Lepidoptera: Geometridae), biological control agents of the weed Mimosa pigra Tim A. Heard a,⇑, Louis P. Elliott b, Bron Anderson b,1, Laura White b,2, Natasha Burrows b, Arelí Mira a, Ryan Zonneveld a, Gio Fichera a, Richard Chan a, Ricardo Segura c a

CSIRO Ecosystem Sciences, 120 Meiers Rd., Indooroopilly 4068, Australia Weed Management Branch, Department of Natural Resources, Environment, the Arts and Sport, Northern Territory Government, P.O. Box 496, Palmerston, NT 0831, Australia c CSIRO Ecosystem Sciences, Mexican Field Station, A. Carlon No. 5, Col. Ejido 1 de Mayo Boca del Río, C.P. 94297 Veracruz, Mexico b

a r t i c l e

i n f o

Article history: Received 26 May 2010 Accepted 9 August 2010 Available online 14 August 2010 Keywords: Woody weed Aquatic weed Legume Biological control of weeds Invasive plants

a b s t r a c t Macaria pallidata (Warren) and Leuciris fimbriaria (Stoll) (Lepidoptera: Geometridae) are abundant and damaging defoliators of Mimosa pigra L. in the native range of the Neotropics. Both species were assessed for their suitability as biocontrol agents of M. pigra, a damaging invasive weed of northern Australia. Larvae feed on leaves of all ages. Adults are non-feeding, short-lived moths. Generation times are short and fecundity is high allowing rapid population increase. The host specificity of these species was tested using larval development tests on 70 test plant species. Development to adult of M. pallidata occurred on six species other than M. pigra. However, the survival rates were so low that these plants could not sustain a population of this insect species. The maximum survival rate was 1.1% compared to 64% on M. pigra. When the mean lifetime fecundity is considered, a survival rate of 1.1% is the minimum required for population maintenance in the absence of other mortality factors. Open-field trials in Mexico, although not comprehensive, support the conclusion that M. pallidata is specific to M. pigra. Development beyond first instar of L. fimbriaria did not occur on any species other than M. pigra and Mimosa asperata L. The number of eggs laid by M. pallidata was independent of plant phylogeny, but adults of L. fimbriaria laid more eggs on plants more closely related to M. pigra, indicating that not all Lepidoptera show indiscriminate oviposition choices in confined situations. Following the gaining of required permits, M. pallidata was released from 2002 and L. fimbriaria from 2004. Both have established. M. pallidata has been recovered in large numbers from most sites across areas of infestation of the invasive plant, but experiences extreme population fluctuations. L. fimbriaria has been found only at low levels. The rate of parasitism of M. pallidata was 5% with the tachinid fly Carcelia malayana Baranov being the most common parasite. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Mimosa pigra L. (Leguminosae: Mimosoideae) is a prickly, perennial, woody shrub native to tropical America from Mexico to Argentina. Outside its native range, especially Australia and South East Asia, it is a highly invasive and damaging weed of wetlands, affecting agriculture and conservation. It forms ⇑ Corresponding author. Fax: +61 7 3214 2885. E-mail addresses: [email protected] (T.A. Heard), [email protected] (L.P. Elliott), [email protected] (B. Anderson), [email protected] (L. White), [email protected] (A. Mira), [email protected] (R. Zonneveld), gio. fi[email protected] (G. Fichera), [email protected] (R. Chan), csiromfs@prodigy. net.mx (R. Segura). 1 Present address: Western Australian Department of Environment and Conservation, Frankland District Office, Australia. 2 Present address: WetlandCare Australia, Ballina, NSW, Australia. 1049-9644/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2010.08.001

impenetrable near monospecific thickets (Lonsdale, 1992) that, compared to natural areas, have a greatly reduced diversity of plants and animals (Braithwaite et al., 1989). This weed competes with pastures, hinders mustering of livestock, and restricts access to water by people and livestock. In Australia, it covers an area of over 800 km2 in the Northern Territory. In 2002, an infestation was discovered in Queensland (Chopping, 2004) and another in 2009 in Western Australia. In 1999, it was declared one of Australia’s 20 weeds of national significance (Thorp and Lynch, 2000). Major efforts in both integrated management (Paynter and Flanagan, 2004) and biological control (Heard and Paynter, 2009) have helped to ameliorate the M. pigra problem. Twelve biological control agents, including ten insects and two fungi, had been released in Australia prior to the agents that are the subject of this paper (Heard and Paynter, 2009). These agents integrate well with other means of control such as fire, herbicides, and mechanical

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clearing (Paynter and Flanagan, 2004). The impact of the four most effective agents: Acanthoscelides puniceus Johnson, Coelocephalapion pigrae Kissinger, Neurostrota gunniella (Busck) and Carmenta mimosa Eichlin and Passoa, especially the latter, has been to reduce seed rain and soil seed banks by up to 90% (Paynter, 2005). Percentage cover of competing vegetation (principally grasses) was significantly higher under stands defoliated by C. mimosa, inhibiting M. pigra seedling establishment and apparently increasing the susceptibility of M. pigra to fire, by increasing fuel loads beneath stands. Four of the eight stands where C. mimosa was absent expanded and none retreated. In contrast, none of the nine stands where C. mimosa was present expanded and three retreated. Analyzing the age-structure of M. pigra stands indicated that retreating stands were typified by a reduction in seedling density (Paynter, 2005). C. mimosa is still spreading into new sites and has become denser at existing sites (Ostermeyer and Grace, 2007). However, it is clear that adequate control has not yet been achieved. Recently, models have indicated that the reduction in fecundity is not the most important impact of biocontrol of M. pigra; rather, it is through defoliation at the edges of stands allowing grasses to out-compete M. pigra seedlings (Buckley et al., 2004). In this paper, we present data on the biology and host specificity of two defoliators, Macaria pallidata and Leuciris fimbriaria and describe the efforts to rear, release and assess their establishment. M. pallidata (Warren, 1897) (Lepidoptera: Geometridae: Ennominae: Macariini) is extremely widespread across tropical America (based on the material in the Natural History Museum, UK (M. Scoble pers. comm.)). It is also recorded (as Xenoecista pallidata, its junior synonym (Scoble and Krüger, 2002)) from Arizona, USA (Hodges et al., 1983). L. fimbriaria (Stoll, 1781) (Lepidoptera: Geometridae: Ennominae: Cassymini) is distributed in Central and South America (Pitkin, 2002). A survey of natural enemies of M. pigra recorded both species from the Mexican States of Guerrero, Oaxaca, and Veracruz (Harley et al., 1995). No host records were found in the literature for either species. Geometridae have been used successfully in weed biocontrol programs. Comostolopsis germana Prout damages shoot tips of bitou bush, Chrysanthemoides monolifera (L.) Norlindh, in Australia and populations at some sites are well established and causing reduction in seed set of up to 70% (Adair and Edwards, 1996). Aplocera plagiata L. established and is occasionally effective against St. John’s wort (Hypericum perforatum L.) in Canada and USA, but not in Australia (Julien and Griffiths, 1998). Several species of Geometridae from Africa have been released in Australia against Acacia nilotica (L.) Willd. ex Delile. Chiasmia inconspicua (Warren) was first released in 1998 and Chiasmia assimilis (Warren) in 2000. C. assimilis, but not C. inconspicua, established quickly and spread rapidly in coastal areas of Queensland (Palmer et al., 2007).

2. Materials and methods 2.1. Diversity and breeding M. pallidata was identified by M. Scoble (The Natural History Museum, UK). The early identifications of L. fimbriaria were provided by D.C. Ferguson (Systematic Entomology Laboratory, USDA) and were confirmed by genitalia dissections of specimens from the laboratory colony by L. Pitkin (The Natural History Museum, UK). Voucher identified specimens of both species are kept at the Australian National Insect Collection. Three shipments of larvae of M. pallidata from Veracruz State, Mexico, were received into quarantine in Brisbane, Australia in 1998 and 2000. Seven shipments of larvae and pupae of L. fimbriaria from Veracruz State, Mexico, were received at the same facility between 2001 and 2004. Insectary colonies of both species

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provided abundant adults for study. Larvae were reared on potted M. pigra plants in aluminum frame cages lined with gauze and measuring 250  250  800 mm. More plants were added to the cages as needed while the larvae developed. Emerging adults were collected approximately 2–3 times per week and added to clean cages with fresh plants in batches of approximately 20 pairs which mated and laid eggs on the plants to start the next generation. Care was taken to mix the adults of each shipment by pairing females from one shipment with males from another to ensure maximum likelihood of maintaining genetic diversity in the cultures by minimizing inbreeding, especially sibling mating, and avoiding potential laboratory adaptation. Only the tested genotypes from sites in Veracruz were released in Australia. 2.2. Biology To determine duration of the life cycle, cohorts of eggs were collected and reared until adults. To determine fecundity and longevity of adults, newly-emerged adults (<24 h old) were collected and sexed. One female and two males were held within a plastic bag over the tip of a live M. pigra plant. Each day the adults were transferred to a fresh tip. Mortality was noted and the number of eggs deposited onto the tips was counted. To determine egg viability, 10 collections of approximately 50 eggs each were made randomly, the eggs were allowed to develop on leaves until the eggs changed color, then transferred to petri dishes where the percentage that hatched was recorded. To determine the emergence pattern of adults, the rate of emergence of both sexes from groups of equal age larvae was recorded. 2.3. Host specificity testing Larval development on selected plant species was used as the primary indicator of the host specificity of these insect species. Adult oviposition preferences could not be used to indicate field host range because females of both species oviposit almost indiscriminately on most surfaces when confined in cages, including most plant species and cage walls. Note however that L. fimbriaria never oviposited on some plant species which were then considered unacceptable to ovipositing females and hence no larval development trials were needed. For M. pallidata, a limited number of plant species were also tested in an open-field trial in Mexico, in which an attempt was made to determine oviposition acceptability and larval development suitability under natural conditions. Adult feeding specificity was not tested as adults of both M. pallidata and L. fimbriaria, like most Lepidoptera, do not feed destructively or do not feed at all. 2.3.1. Laboratory larval development tests Adults were placed in an inflated clear plastic bag measuring 500  300 mm enclosing the tip of the plant. Females avoid oviposition on the smooth plastic surfaces, so the use of the plastic bag encouraged oviposition directly onto the plant. The adults were 1–3 days old when placed on the plants having been left in their breeding cages following emergence, to allow opportunities to mate before collection and use. Two tips per plant were used. For M. pallidata, three pairs of adults were enclosed in each bag, requiring six pairs of adults per plant per replicate. For L. fimbriaria, one tip held three pairs of adults and the other held five, therefore eight pairs of adults were used per plant per replicate. After 3 days for M. pallidata and 4 days for L. fimbriaria, the adults and bags were removed from each plant tip. The eggs, which are laid on both sides of the leaves, on the stems and in the nodes, were counted and recorded. One M. pigra control plant and a variable number of test plants were used for each trial. For validity, the fecundity of adults and

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viability of the immature stages had to be confirmed. For fecundity, >40 eggs had to be laid on each plant replicate. For immature stage viability, the proportion of the eggs that resulted in emerged adults on M. pigra had to exceed 30% for M. pallidata and 10% for L. fimbriaria. When excessive numbers of eggs were laid on the M. pigra control plant, some were removed to avoid larval competition for resources, resulting in a maximum of 425 eggs for M. pallidata and 588 for L. fimbriaria. All eggs were left on the test plants because survival during the early-instars was low and competition was never a concern. Adults of L. fimbriaria never oviposited on some plant species. If no oviposition occurred on a species after four replicates, the test was discontinued. These plant species were considered unacceptable to ovipositing females and hence no larval development trials were needed. Other species had eggs laid on them occasionally and in low numbers. These plants were considered potential hosts on the basis of oviposition, but had insufficient eggs to test larval development reliably. For each plant species, different individual plants were used for each replicate. Initial studies showed that leaves of M. pigra of all ages are suitable for larval development and so no special plant requirements were needed concerning leaf age. The plants were held for larval development in an aluminum frame cage lined with gauze and measuring 250  250  800 mm or 250  250  500 mm depending on the size of the plant. They were monitored regularly and extra plants of the same species were added if the larval feeding depleted the original plant. All plants were held for 45 days, by which time all adults had emerged from the M. pigra control plant (most had emerged by 35 days, but extra time was allowed in case development was slower on test plant species). At this date, all test plants were examined for larvae and signs of larval feeding. For M. pallidata, a further series of replicates were done on Acacia decurrens Willd. and Neptunia dimorphantha Domin, the two species that (with the exception of Mimosa asperata) recorded the greatest numbers of developed adults. In these trials, plants were saturated with eggs. Five replicates that each consisted of five pairs of adults placed in bags on plant tips were completed for each species. This allowed a more precise measure of survival rates and increased the opportunity of obtaining mating pairs to check the viability of females. The test plant list for this target weed has been approved for many years (Forno and Heard, 1997). The list has been modified for each agent for various reasons including: the known hosts of close relatives of the agent, the particular features of the biology of the agent and changes to our understanding of the systematics of the legume families. For M. pallidata, L. fimbriaria and other leaf-feeding agents, this has been increased to include representatives of the Botrycephalae, the section of the Acacia genus with pinnate (rather than phyllodinous) leaves which are morphologically similar to those of M. pigra. The list used for testing this insect consists of 70 (69 for L. fimbriaria) species. Such a long list was considered necessary due to the size, diversity and importance of the legume plant families. 2.3.2. Relationship between plant phylogeny and oviposition by M. pallidata and L. fimbriaria Data from the laboratory larval development tests on M. pallidata and L. fimbriaria was used to investigate the relationship between plant phylogeny and oviposition acceptance. 2.3.3. Open-field trial of M. pallidata in Mexico This trial was done in a 1000 m2 plot of land in a rural area near the city of Veracruz, Veracruz State, Mexico. A total of 12 plots were each planted with four representatives of four of the test species; each plot included M. pigra. Not all plants thrived under the conditions but 28 mainly Australian species were sufficiently

healthy to be included in the data recording. Between 17 August 2000 and 2 February 2001, a total of 622 laboratory-reared adults of M. pallidata were released in 12 batches. When larvae appeared on plants, the leaves were enclosed in a gauze bag to capture the emerging adult for identification. To quantify the suitability of Neptunia plena (L.) Benth, an oviposition and larval development test on this species with a M. pigra control was done in the laboratory garden of the CSIRO Mexican Field Station. Two pairs of adults were added to a bag covering a living plant growing in a pot. Six replicates of both species were completed. Eggs were counted and the resulting larvae were allowed to feed and develop on the plants. The emerging adults were counted. 2.4. Mass-rearing and release Mass-rearing was conducted in a manner similar to the rearing in the quarantine laboratory described in Section 2.1, except several refinements were made. Before use, potted plants were submerged in water for a day to remove any ants or other pests that might be living inside the pot. In the dry season, measures were taken to prevent desiccation of the eggs: sheets of plastic were attached to the sides and the tops of the cages and a mist of water was sprayed into the cage every day until most of the larvae had hatched. A piece of material was attached from the stem of the plant to the outside of the pot to prevent pupation in the soil in the pots as pupae are more easily collected from the folds in the gauze than from the soil. Towards the end of the larval stage, several petri dishes of vermiculite were placed around the cage for the insects to pupate in as it was easier to collect the pupae from vermiculite than from the bottom of the cage. Once they had pupated, the remaining foliage was removed to make it easier to catch the emerging adults. The first adults to emerge were taken to start the next generation; the remainder were collected for release. Males and females from different cages were crossed. Fresh genetic material from Mexico was occasionally used to start new colonies to reduce the risk of inbreeding or laboratory adaptation. Larvae, pupae and adults were released but the preferred method was to place pupae in a 6 L, Styrofoam container which allowed adults to disperse in as natural a manner as possible following eclosion on site. Each container had a mesh lid with purposefully-cut holes (8 mm diameter) which were large enough for the adults to escape but small enough to exclude predators such as frogs and lizards. A piece of damp foam was placed in the bottom of the container to prevent the pupae from desiccating. The pupae were placed in a 1 L plastic container within the 6 L Styrofoam container which was sealed with tape and suspended from a M. pigra branch by string. A waterproof metal cover was attached to the string to prevent rain from penetrating the container and to provide shade. A sticky gel was applied to the string and the M. pigra branch to prevent ants entering the container. The advantages to this method of release are: the delicate adult stage does not need to be transported or handled and it is less time consuming to collect pupae than it is to catch and release adults. In addition to the release of pupae, some larvae were released directly onto plants. Adults were also released, usually at night, when they were active and fewer bird predators were present. 2.5. Evaluation of establishment 2.5.1. Survey of presence of M. pallidata at multiple sites, 2003–2004 In May 2003, M. pallidata was monitored at 13 sites. All of the original release sites were revisited and a visual search was undertaken. In May 2004, M. pallidata was searched for as part of a comprehensive survey, across the distribution of M. pigra in the Northern Territory, for seven of the 13 M. pigra biocontrol agents

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(Ostermeyer and Grace, 2007). In total, 87 sites were surveyed covering the Adelaide, Finniss, Mary, Daly and Moyle River catchments (Fig. 1). These sites were chosen from detailed M. pigra distribution data collected in 2003 (C. Yates, unpub. data). Each site was surveyed for M. pallidata presence regardless of whether it had been released there or not. The search was conducted by inspecting M. pigra leaves for the presence of larvae for 10 min (normally 5 min two people). Any larvae or adults found were collected in small clear plastic containers and taken back to the laboratory where they were placed in bags on potted plants to complete development to adult for identification. Before being used for rearing larvae all potted plants were washed and searched for larvae, as a population of M. pallidata was established on the M. pigra plants growing at the laboratories. Because other geometrids have been recorded on M. pigra (Wilson et al., 1990) all adults obtained were pinned and their identity was confirmed.

2.5.2. Monthly surveys of abundance of geometrid larvae at six sites, 2005–2010 (visual survey and beat samples) From September 2005 until March 2010 abundance of M. pallidata and L. fimbriaria was measured at six sites monthly between 2005 and 2008, reduced to 4–5 times per year between 2009 and 2010. Typically access was impossible during the peak of the wet season between February and April and hence surveys could not be made in these months. Visual surveys were done in the same way as for the 2003–2004 surveys described above, except that the larvae were counted, not just recorded as present or absent. Beat samples were also taken at these sites; 40 tips were shaken into a 250 mm wide funnel attached to a collection jar containing 70% ethanol. Geometrid larvae in the samples were counted on return to the laboratory. Although it requires rearing to adult stage to be certain of identity, experienced observers can readily distinguish L. fimbriaria larvae from M. pallidata in the field, principally by the presence of well-defined longitudinal pale stripes.

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2.6. Parasitism To determine the abundance and diversity of parasites, larvae were collected and reared for the emergence of either adults or parasitoids. The larvae were collected from eight sites on four river systems between March 2006 and June 2009. In total there were 57 collections made which yielded a total of 327 larvae with between 1 and 18 larvae per batch. 3. Results 3.1. Biology Both species present similar biology. Eggs are laid onto stem or leaf surfaces, often on hairs or on the margin. Larvae begin feeding by removing the top surface of the leaf. Larvae feed exposed on young and mature foliage. Larvae drop on a silken thread when disturbed. They move very little from their original position until forced to look for more food. They are active at night. During the day they remain stationary in one of two positions: either holding the substrate with the prolegs and with the remainder of the body straight to mimic a twig, or with both the prolegs and legs in contact with the substrate and the body in an arched position. Pupation is in the soil or among damaged plant tissue. Adults are nocturnal and feed on nectar sources. They are very common and damaging insects on M. pigra in the native range in Mexico. Both are multivoltine species which occur throughout the year in the native range but are more common in the rainy season. The mean duration of the life cycle of M. pallidata from egg to adult is 25 days, consisting of 5, 14 and 6 days for the egg, larval and pupal stages, respectively. Five larval instars were identified based on head capsule widths. The average lifespan for females was 5 days (n = 21, range: 3–6) and for males was 5.5 days (n = 30, range: 1–8). Females laid a lifetime mean of 182 eggs, (n = 21, range: 0–318, SE = 25). Females began laying eggs on the

Fig. 1. The 87 sites at which Macaria pallidata was surveyed for presence or absence in August 2004. Known release sites that occurred prior to this survey are also shown, except for one release on the Moyle River catchment (location unknown) believed to have taken place in October 2003.

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first night and continued until death. A mean of 75% of eggs hatched (n = 10, range: 0–100%, SE = 9%). If the one zero value which probably represented a failure to mate was excluded, this changes to mean of 84% of eggs hatched (n = 9 range: 56–100%, SE = 4%). The mean duration of the life cycle of L. fimbriaria from egg to adult is 28 days, consisting of 5, 18 and 5 days for the egg, larval and pupal stages, respectively. Females laid a lifetime mean of 90.2 eggs, (n = 12, range: 0–217, SE = 20.5). The average lifespan for females was 4.5 days (n = 12, range: 2–7) and for males was 3.6 days (n = 12, range: 1–6). Females began laying eggs on the second night and continued until death (Fig. 2). The emergence of L. fimbriaria males and females is synchronous (Fig. 3). The sex ratio is approximately 50:50 (Fig. 3). In laboratory conditions, nectar sources did not appear to enhance the survival of adults of L. fimbriaria and so this species may not naturally feed in the adult stage. 3.2. Host specificity testing

12

50 45 40 35 30 25 20 15 10 5 0

Eggs/female/day Survival

10 8 6 4

Survival

Eggs per female per day

3.2.1. Laboratory larval development tests of M. pallidata A total of 16,170 eggs were laid on the 52 replicates of M. pigra plants with 64% surviving to adulthood. The mean number of eggs laid on the test plant species combined was 179. Although more (312) were laid on M. pigra, it is clear that adults will accept non-hosts for oviposition when confined in cages. Of the 50,530 eggs laid on 70 test plant species, only 46 survived to adults and these on only six species of plants: M. asperata (an exotic species to Australia) (27 adults), N. dimorphantha (7), A. decurrens (7), Acacia oshanesii F. Muell. and Maiden (2), Acacia irrorata Sieber ex Spring (2), and Acacia parramattensis Tind. (1) (Supplementary material, Table 1). The adults which emerged from the test plant species often were deformed. The viability of females could not be tested as pairing of females with males was not possible because the few adults that emerged did so asynchronously over a prolonged period.

2 0 1

2

3

4 5 Day from emergence

6

7

8

Fig. 2. Age specific survival and fecundity (mean ± SE) of females of Leuciris fimbriaria.

No. of adults emerged

300 250

females

200

males

150

For the further series of replicates on A. decurrens and N. dimorphantha, a total of 4128 eggs were laid on N. dimorphantha with only 28 adults developing. This equates to a mean of 0.63% survival which is 100 times lower than the 60.8% recorded on M. pigra. From these 28 adults, seven pairs were set up on tips for mating and oviposition. The mean fecundity of the seven females was 44 eggs (SE = 32.9) showing that they were fertile, although less so than females bred from M. pigra. Only five second-generation N. dimorphantha-bred adults emerged and four of these were deformed. Survival on A. decurrens was even lower with only five adults developing from a total of 3771 eggs. This equates to 0.12% survival compared to 59.2% recorded on the M. pigra control. Of these five adults, three were males and all of these were deformed. Hence it was impossible to verify the fecundity of the females. 3.2.2. Open-field trial with M. pallidata in Mexico In the open-field trial in Mexico, a total of 31 adults emerged from the plants (Supplementary material, Table 2). Only two of these were from plants other than M. pigra: one from each of M. asperata and N. plena. Both of the adults were deformed. N. plena is a native Mexican species, unlike the other tested Neptunia species which are Australian natives. It is notable that the Australian Acacia and Neptunia species which supported larval development in laboratory trials were not attacked in the field. While this trial provides some useful supporting information, we do not consider it to be conclusive in its own right because of the low level of attack on the M. pigra plants. Although 622 adults were released, only 31 next generation adults were reared from plants in the plot. This indicated that females were either dying or not remaining in the plots to oviposit on the plants there. The oviposition and larval development test resulted in more eggs being laid on M. pigra (mean = 358, n = 6, SE = 60) than on N. plena (mean = 181, n = 6, SE = 33). Adults only emerged on M. pigra (mean = 94, n = 6, SE = 29) with none developing on N. plena. 3.2.3. Laboratory larval development tests of L. fimbriaria A mean of 232 eggs (total = 9522, SE = 18, range = 53–588) were laid on M. pigra control plants in the 41 valid trials (Supplementary material, Table 3). A mean of 68% of these developed to adults. A grand total of 15,431 eggs were laid on the 68 test plant species. With the exception of one plant species, none of these eggs developed through to adults (Supplementary material, Table 3). Observations on a subset of these eggs showed that they hatched but failed to commence feeding and died in the first larval stage. The exceptional species was M. asperata, on which a mean of 183 eggs (total = 732, SE = 19, range = 145–223) were laid of which a mean of 77% developed through to adults. 3.2.4. Relationship between plant phylogeny and oviposition by M. pallidata and L. fimbriaria The number of eggs laid by M. pallidata was statistically independent of plant phylogeny (Table 1); although there were slightly fewer eggs laid on more distantly related species, this was not significant (v2 = 7.7, P = 0.10). In contrast, the number of eggs laid by L. fimbriaria was not random but followed plant phylogeny (Table 1). The mean number of eggs laid on plants decreased as the relatedness decreased (v2 = 46.2, P < 0.001). Adults never oviposited on 12 of the plant species.

100

3.3. Mass-rearing and release

50

40

37

34

31

28

25

22

19

16

13

10

7

4

1

0 Days from egg lay Fig. 3. Emergence of females and males of Leuciris fimbriaria from time of egg lay.

The relative numbers of the various stages released, the timing and duration of the releases and the river catchments in which releases were made are shown in Tables 2 and 3. The relative locations of the river catchments are indicated in Fig. 1.

T.A. Heard et al. / Biological Control 55 (2010) 248–255 Table 1 The number of eggs laid by Macaria pallidata and Leuciris fimbriaria on plant species as a function of their relatedness to Mimosa pigra. Phylogenetic distance

No of species tested M. pallidata/L. fimbriaria

Same genus Same tribe, different genus Same family, different tribe Same order different family Different order

Mean eggs laid per plant Macaria pallidata

Leuciris fimbriaria

4 10/9

269 219

230 58

25/24

201

52

19

131

32

13

153

11

Table 2 Timing and numbers of the various stages of Macaria pallidata released. River catchment

Period of release

Larvae

Pupae

Adults

Total released

Adelaide Daly Finniss Moyle

06/2002–05/2004 10/2004–12/2004 08/2002–10/2004 10/2003

97 10 801 0

4167 2000 12,760 0

5355 842 11,406 277

9619 2852 24,967 277

Total

908

18,927

17,880

37,715

Table 3 Timing and numbers of the various stages of Leuciris fimbriaria released. River catchment

Period of release

Larvae

Adelaide Daly Finniss Mary Moyle

12/2004–03/2008 09/2006 07/2005–11/2006 02/2006–08/2006 09/2006

3150 0 0 0 0

Total

3150

Pupae

Adults

Total released

9532 80 9460 1432 2000

10,348 120 5043 550 100

23,030 200 14,503 1982 2100

22,504

16,161

41,815

3.4. Evaluation of establishment 3.4.1. Survey of presence of M. pallidata at multiple sites, 2003–2004 In May 2003, within one year of release, M. pallidata was found at three out of the 13 sites surveyed. In August 2004, M. pallidata was found at 44 out of the 87 sites surveyed (Fig. 1) including sites in the Daly and Mary River catchments where it had not been released prior to the survey. In the Mary River catchment, the furthest site where M. pallidata was found from its nearest release site was approximately 52 km, evidence of considerable mobility throughout the landscape. The survey sites were representative of the overall distribution of high density M. pigra in the area shown in the map (Fig. 1). All geometrid larvae found were confirmed as M. pallidata. 3.4.2. Monthly surveys of abundance of geometrid larvae at six sites, 2005–2009 (visual survey and beat samples) The highest peaks in M. pallidata larval abundance were detected between January and May, which broadly corresponds to the wet season in northern Australia (Fig. 4). The same pattern was evident across all three catchments surveyed, however the maximum number of larvae detected was very different between catchments (maximum larvae detected in visual survey: Mary = 152; Adelaide = 30; Finniss = 7; maximum larvae detected in beat survey: Mary = 126; Adelaide = 15; Finniss = 9).

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In February 2007 (and to a lesser extent, February 2006), the Mary River sites experienced a population explosion of M. pallidata which caused widespread and massive defoliation. Since this time, the sites on the Mary River have been so severely affected by a dieback pathogen (currently under investigation), that these sites are unlikely to support a significant population of leaf-feeding agents for the immediate future. The wet seasons of 2006 and 2007 were good years for M. pallidata, in contrast to the poorer years 2008– 2009 in which few larvae were found. An increase in abundance appeared to be occurring in 2010 in the Finniss and Adelaide catchments. No L. fimbriaria were recovered in this survey. 3.4.3. Opportunistic sightings of L. fimbriaria Despite not being able to detect L. fimbriaria larvae in the course of the monthly abundance surveys, L. fimbriaria larvae were found in the process of collecting M. pallidata larvae from the Adelaide River catchment for the purposes of detecting parasitoids (one individual in April 2009) and for establishing new colonies (six individuals). Of the six larvae detected while establishing new colonies, two individuals were detected on two separate occasions in March and April 2009, and four individuals were detected from two occasions in March 2010. Their identity was confirmed by rearing them through to the adult stage. On each occasion, L. fimbriaria comprised approximately 10% of the total number of larvae collected. This proportion would not be representative of the landscape as a whole, as the larvae were collected from only one site in the Adelaide River catchment. In addition, one L. fimbriaria adult came to a light trap at Melaleuca Station (Mary River) in September 2009, and a larva found on the potted M. pigra plant at Berrimah Research Farm (Darwin) in 2009 proved to be L. fimbriaria. It is possible that a low proportion of larvae counted in the monthly M. pallidata surveys (Fig. 4) were actually L. fimbriaria. These records show that L. fimbriaria is established broadly in the landscape (in the Mary and Adelaide River catchments), but at low abundance, certainly much lower than that of M. pallidata. 3.5. Parasitism From the 327 larvae collected, 226 (69%) developed to adult. Only one adult of L. fimbriaria emerged, the remaining 225 were M. pallidata. 17 parasitoids emerged, giving a mean of 5% parasitism. The remaining 84 larvae (26%) either died or went missing. Of the 17 parasitoids, 14 were the Carcelia malayana (Tachinidae) and three were identified as Glyptapanteles sp. (Braconidae) by A. Austin (University of Adelaide), however as no female specimens were found, the species determination of Glyptapanteles cannot be made. 4. Discussion The results of the host specificity testing showed that both insect species are sufficiently specific to be acceptable for release in Australia. The survival rate of M. pallidata on all plant species other than M. pigra was too low for any of these species to support a population. To maintain a population, the minimum production of adults per female must be a minimum of two; which, at a mean fecundity of 182 eggs per female observed in the life history studies, requires a larval survival rate of 1.1%. The highest survival rate obtained was 1.1% on A. decurrens. Hence, even in the unlikely scenario where no other mortality factors exert pressure, populations of this insect could not increase on this host. The yield of an adult in the open-field trial in Mexico from N. plena indicates that this species could be a real host in the native range, although no development was found in further tests. As other species of Neptunia occur naturally in northern Australia (one species, Neptunia major (Benth.) Windler, is sympatric with M. pigra), there is a possibility

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Fig. 4. Average number of Macaria fimbriaria larvae on three river catchments counted by (a) visual survey, and (b) beat survey.

that these plant species may experience some oviposition and low levels of larval damage. This possibility of non-target impact needs to be monitored. Larval development of L. fimbriaria only occurred on two plant species, the target weed, M. pigra and M. asperata. Mimosa asperata (=Mimosa pigra var. berlandieri) is a close relative of M. pigra (Barneby, 1991). It does not occur in Australia but was grown under quarantine and used to aid the circumscription of the host range of potential biocontrol agents. L. fimbriaria is a common and widespread species in Trinidad and has been reared there from Mimosa by M. Yaseen (M.J.W. Cock, pers. comm.) most probably from Mimosa pudica L. as this is the species surveyed and M. pigra is very localized on the island. Our tests showed that the population from Mexico was unable to develop on M. pudica. This suggests geographic variation in host use by L. fimbriaria. In any case, all field

evidence indicates that the host range is confined to the Mimosa genus. The number of eggs laid by M. pallidata in the larval development tests was independent of plant phylogeny indicating a lack of oviposition discrimination in the laboratory by this species. In contrast, the number of eggs laid by L. fimbriaria is not random but follows plant phylogeny. This suggests that adults of this species are not just ‘‘dumping” eggs on any substrate but respond to cues presented by the plant. As the relatedness decreases, the number of common cues also decreases, the plant is less attractive, and fewer eggs are laid (Weiblen et al., 2006). This demonstrates that not all Lepidoptera show indiscriminate oviposition choices in confined situations. Large numbers of both species were mass-reared for release, over 37,000 M. pallidata between 2002 and 2004 and over 41,000

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L. fimbriaria between 2004 and 2008. M. pallidata established quickly and has demonstrated an ability to spread to an adjacent river catchment. This has been the fastest dispersing insect on M. pigra, with the possible exception of N. gunniella (Wilson and Flanagan, 1990). Populations of M. pallidata were generally low, but were consistently present and on occasions reached very high numbers. On these occasions, plants were largely stripped of their green leaves. Defoliation of M. pigra plants by larvae of M. pallidata significantly reduces growth rate and plant height (Wirf, 2006). Population peaks of M. pallidata are linked to wet season rainfall when plants are of better quality. In the area of this study, the wet season starts in December and a dense canopy develops within a month (Lonsdale, 1992). This area experiences a particularly severe dry season and M. pigra responds to water stress by shedding large quantities of leaves (Lonsdale, 1988). In the Mary River catchment, very high numbers of M. pallidata larvae were observed in February 2006 and 2007 only. However, in the Finniss and Adelaide catchments, more consistent numbers of M. pallidata larvae were recorded in the period between February and May across all years. M. pallidata populations need one to two months following the production of fresh plant growth to build up numbers. The host plant does not provide a good food source in the late dry season for a species with an obligate leaf-feeding stage. Recent wet seasons have not shown the peaks of M. pallidata abundance observed in 2006 and 2007. We suggest a number of potential contributing factors: (1) parasitoids have been observed to attack the larval stage of this insect; (2) ants and birds have been observed to disturb and reduce numbers of M. pallidata larvae (Grace, 2005); (3) inter-year differences in the onset and departure of the wet season may provide good conditions for M. pallidata in some years, but not others. Parasitoids alone are unlikely to result in low levels of M. pallidata in the field because the rate of recorded parasitism is only in the order of 5%. Predators may be an important factor in reducing numbers but probably would not result in the strong seasonality observed of M. pallidata abundance. This leaves the phenology of its food source as an important factor in M. pallidata abundance. Further monitoring of M. pallidata populations will provide a clearer description of its establishment and impact on M. pigra. Acknowledgments Moises Martinez provided able technical assistance in Mexico. John Scott, Geoff Baker and Mic Julien (CSIRO) reviewed a draft. Marianne Horak, CSIRO identified the Australian collected adults. Funding from CSIRO, the Environment Australia, through the Natural Heritage Trust, Weeds of National Significance program supported this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biocontrol.2010.08.001. References Adair, R.J., Edwards, P.B., 1996. An attack strategy against Chrysanthemoides monilifera, a weed of native vegetation in Australia. In: Moran, V.C., Hoffmann, J.H. (Eds.), Proceedings of the Ninth International Symposium on

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