**Utilization of the exotic weed Pluchea odorata (Asteraceae) and related plants by the introduced biological control agent Acinia picturata (Diptera: Tephritidae) in Hawaii**

Andrei V. Alyokhin, Russell H. Messing, and Jian J. Duan

This is an electronic version of an article published in Biocontrol Science and Technology (Volume 11, Pages 703-710). Biocontrol Science and Technology is available online at: http://www.informaworld.com. Reproduced with permission of the publisher. Please send me an e-mail if you want a PDF reprint of this article in its original journal format.

ABSTRACT

The flower-head feeding fly Acinia picturata (Diptera: Tephritidae) was deliberately introduced from Mexico into Hawaii in 1959 for biological control of the exotic weed Pluchea odorata (Snow) (Asteraceae). Neither field efficacy nor possible non-target effects of the fly have been evaluated in the 40 years since the introduction. We assessed the impact of the fly on both its target host and on 7 non-target plant species. The impact on the target weed was limited, with only 5 - 13% of the developing seeds in P. odorata flowerheads being destroyed by larval feeding. We did not detect any host range expansion of A. picturata onto flowerheads of 2 exotic or 5 endemic non-target plant species in the family Asteraceae.

Key words: classical biological control, weeds, Acinia picturata, Pluchea odorata, host specificity, non-target impacts, endemic plant species

Introduction

Introductions of herbivorous insects have been successfully used to suppress exotic weeds both in agricultural and natural settings, with approximately 20% of all introductions resulting in significant control of the target plant species (Andres et al., 1976; De Bach & Rosen, 1991; Van Driesche & Bellows, 1996; Williamson & Fitter, 1996). Historically, a majority of scientists and the general public viewed classical biocontrol as an efficient, low-cost, and environmentally safe approach to pest management (De Bach & Rosen, 1991). Most of the early insect introductions for biological control of weeds involved the release of many herbivorous species after only limited host range testing (Harris, 1998). Fairly soon, however, the safety of introducing untested plant feeding insects was questioned, resulting in the establishment of host-screening requirements for potential biocontrol agents. At first, the major focus of host-testing protocols was on possible damage caused by released agents to economically important crops. Later, testing was expanded to include native plant species thought to be at risk (Harris, 1998). Finally, a comprehensive scientifically-based protocol for testing herbivorous insects prior to their release in new areas was developed (Wapshere, 1974, 1989). This protocol is currently widely used by biocontrol practitioners; however, the cost of its implementation may be as high as $580,000 per insect species under consideration (Harris, 1998). It is likely that in the near future regulators will require even more thorough risk-benefit analyses for all prospective biocontrol agents, and that releases will be permitted only when the benefits of introduction are expected to significantly outweigh the risks to non-target species.

Despite fairly rigorous evaluations of potential non-target effects caused by new biocontrol agents, the use of exotic herbivores to suppress weed populations continues to create controversy among ecologists, conservation biologists, and biocontrol practitioners (Howarth. 1991; Simberloff & Stiling. 1996; Louda et al., 1997; McEvoy, 1996). A favorite argument used by biocontrol advocates is that there is no convincing evidence of significant adverse ecological effects caused by carefully screened insects released for weed control (Center et al., 1995). However, it is usually countered by the argument that such a lack of evidence is explained by the paucity of follow-up studies, not by the actual absence of ecological damage in the areas of introduction (Howarth, 1991; Simberloff & Stiling, 1996; Louda et al., 1997). Indeed, only rarely have the long-term effects of deliberately introduced exotic herbivores been quantified (Hopper, 1998).

Sourbush, Pluchea odorata (L.) Cass. (Asteraceae), is a fast-growing erect shrub up to 4 m in height, which produces numerous wind-dispersed seeds (Whistler, 1995; Wagner et al., 1999). Native to tropical America, this weed was first reported from Hawaii in 1931. Since then, it has spread to all Hawaiian islands, and it is considered to be a serious problem on Kauai, Oahu, Hawaii, and Maui (Smith, 1985). P. odorata is most abundant in dry habitats, but may also be found in mesic and even wet areas (Whistler, 1995).

In an attempt to control this weed, a flowerhead fly, Acinia picturata (Snow) (Diptera: Tephritidae), was introduced in 1959 from Mexico to the islands of Oahu, Kauai, and Maui (Davis & Krauss, 1962). Within its native range in Guatemala and Mexico, the larvae of this species were known to inhabit P. odorata flowerheads and to feed on its seeds (Davis & Krauss, 1962). After successful establishment in the areas of its release, A. picturata quickly spread throughout the Hawaiian islands (Davis, 1961; Davis & Krauss, 1962). Currently, this fly is quite common in areas invaded by P. odorata (Alyokhin, unpublished). However, little is known about its impact on the plant that it was supposed to control, nor about the possible expansion of its host range to include non-target plants related to P. odorata. In the present study, we investigated the incidence of A. picturata in the flowerheads of target and non-target plant species, and evaluated its impact on seed production by P. odorata.

Materials and Methods

Plant species. Flowerheads of three exotic and five endemic plant species were surveyed for the presence of A. picturata. Exotic species included P. odorata, as well as Elephantopus mollis Kunth (elephant’s foot, tobacco weed) and Sonchus oleraceus L. (sow thistle, pualele). Native Hawaiian species included Bidens hawaiensis A. Gray (ko'oko'olau) and 4 different species of na’ena’e (Dubautia ciliolata Keck, D. scabra Keck, D. laxa Hooker & Arnott, and D. plantaginea Gaudichaud). Because little is known about A. picturata biology and ecology, it was difficult to know what plant species other than P. odorata could be suitable for this fly’s oviposition and development. To account for a broad range of possibilities, we surveyed plants that varied in growth form and habitat (Table 1). However, all of them belong to the family Asteraceae (=Compositae), and overlapped in distribution with P. odorata. If the host range of A. picturata were to expand, these species are the most likely candidates for infestation.

Table 1. Morphological and ecological characteristics of surveyed plant species¹ in Hawaii.

Plant species	Plant type	Inflorescence	Origin	Current distribution	Preferred habitat
Elephantopus mollis Kunth	coarse erect herb up to 1.5 m in height	an open panicle of subglobosal clusters of flowerheads, 1-2 cm wide	tropical America	pantropical	dry, disturbed areas such as plantations and pastures
Pluchea odorata L.	erect shrub up to 4 m in height	a terminal, broad, flat-topped panicle bearing numerous campanulate to cup-shaped heads; involucre 4.5-6 mm long	tropical America	pantropical	common in dry areas, but ranging up to 1000 m in mesic to wet forest
Sonchus oleraceus L.	erect annual herb up to 1.2 m in height	a cyme of 1 to several ovoid heads; involucre 6-10 mm long	Europe	global	disturbed habitats, particularly in croplands and lawns
Dubautia ciliolata Keck	erect, often much branched woody shrub up to 1.8 m tall	elongate, compound panicle 1.5-12 cm long and 1.5-6 cm wide	endemic	island of Hawaii	dry, open habitats from dry shrubland to dry ohia forest, especially on lava, 900-3200 m in elevation
Dubautia scabra Keck	suberect, decumbent, or often mat-forming soft shrub	strongly exerted, compound corymb 2-18 cm long and 2-22 cm wide	endemic	islands of Molokai, Lanai, Maui, and Hawaii	fog-swept, barren, often very recent lava flows or rain forest habitats at 75-2,500 m elevation
Dubautia laxa Hooker & Arnott	shrub 0.5-5 m tall	glomerate-congested corymb 2-17 cm long and 3-27 cm wide	endemic	islands of Kauai, Oahu, Molokai, Lanai, and Maui	wet forests, bogs, and fog-swept ridges, at 360-1,700 m elevation
Dubautia plantaginea Gaudichaud	shrubs to small trees up to 7 m tall	congested, pyramidal, decompound panicles 6-30 cm long and 6-30 cm wide	endemic	islands of Kauai, Oahu, Molokai, Lanai, Maui, and Hawaii	mesic to wet forests and windswept ridges at 300-2,100 m elevation
Bidens hawaiensis A. Gray	an erect perennial herb 0.7-2.5 m tall	compound cymes terminating main stem and lateral branches, 3-5 cm in diameter including ray florets	endemic	island of Hawaii	scattered from open shrubland on old lava flows to mesic forest at 50-1,400 m elevation

¹ Summarized from Whistler (1995) and Wagner et al. (1999).

In Hawaii, flowerheads of E. mollis are attacked by the exotic Tetreuaresta obscuriventris (Loew) (Diptera: Tephritidae), while flowerheads of S. oleraceous are inhabited by the exotic Ensina sonchi (L.) (Diptera: Tephritidae). The first fly species was deliberately introduced to Hawaii from Fiji in 1961 as a part of weed biological control effort (Chong, 1962), and the second fly species was an accidental introduction recorded on the islands for the first time in 1968 (Hardy & Delfinado, 1980). Flowerheads of native Asteraceae in Hawaii are the hosts for a complex of endemic seed feeding Trupanea spp. Schrank (Diptera: Tephritidae) (Hardy & Delfinado, 1980).

Flowerhead survey. Flowerhead samples were collected by excising haphazardly selected inflorescences at approximately bi-weekly intervals between February, 1999 and April, 2000 at a number of different sites on the islands of Kauai, Oahu, and Hawaii. Flowerheads of D. laxa and D. plantaginea, both of which flower during a relatively short period of time, were collected weekly. The exact quantity of sampled material, as well as the location and time of sampling varied depending on plant abundance, distribution and flowering phenology (Tables 2 and 3).
Excised inflorescences were brought to the laboratory, where they were incubated at 24±2°C and natural lighting as described by Duan et al. (1996). Emergence of adult flies was recorded every 3 to 5 days. We felt that removing flowerheads from B. hawaiiensis, thus reducing production of seeds by sampled plants, could possibly affect the already low populations of this species. Therefore, instead of excising inflorescences, we enclosed them in clear nylon-screen bags, and later counted the number of emerged insects directly in the field.

Table 2. Sites of flowerhead surveys in Hawaii, 1999-2000.

Island	District	Location	Elevation (m)	Mean annual precipitation¹ (mm)	Vegetation cover²
Hawaii	Kau	Hawaii Volcano National Park (HAVO)	1000 - 1150	2000-2500	Mixed closed forest with native shrub understory
		Pahala	130	1000	Former sugarcane plantation divided into a number of small vegetable farms
	Hilo	Waiakea Forest Reserve	240-260	5000	Alien closed forest with uluhe-Rubus spp. understory
Kauai	Hanalei	Anini	1-5	1250	Roadside alien weeds
	Kawaihau	Kauai Agricultural Research Center (KARC)	180	1875	Unmanaged open grassland
	Waimea	Kokee State Park	1200	1765	Mixed open forest with uluhe-Rubus spp. understory
	Waimea	Waimea Canyon State Park	1000	1300	Alien closed forest with guava-lantana understory
Oahu	Koolaupoko	Old Pali Rd.	365	3000	Scattered weeds overgrowing abandoned paved road
	Waianae	Kaala Natural Area Reserve	1100 - 1200	2000	Open native forest

¹ Precipitation figures were interpolated from published isohyets based on 67-year rainfall data (Giambelluca et al. 1986).
² Types of plant communities were determined using descriptions and vegetation maps developed by Ripperton and Hosaka (1942), Mueller-Dombois and Fosberg (1974), and Sohmer and Gustafson (1987).

Infestation of P. odorata flowerheads. To control for the possibility of decreased fly survivorship due to experimental handling in the previous study, an additional study was conducted to quantify the population density of immature A. picturata inhabiting P. odorata flowerheads. At each sampling date during the surveys conducted on the islands of Kauai and Oahu in 1999, and on the island of Hawaii in 2000 we haphazardly selected a subsample of 10 flowerheads each from 5 P. odorata plants growing at each of the surveyed sites. These flowerheads were thoroughly dissected under a microscope, and the number of A. picturata larvae and pupae infesting each of them was recorded.

Destruction of P. odorata seeds by A. picturata. Dissections conducted under the previous objective revealed that A. picturata larvae do not consume all the seeds in the flowerhead where they complete their development. To estimate the number of seeds destroyed by a single A. picturata larva, we compared the number of seeds set in 32 flowerheads containing a single fly pupa (but no larvae) to the number of seeds set in another 32 flowerheads collected from the same inflorescences, but containing neither fly larvae nor pupae. The seeds were counted under a dissecting scope. All flowerheads investigated in this study were collected during the survey conducted on the island of Hawaii in 2000.

Insect identification. Flies were identified using the keys developed by Hardy & Delfinado (1980). The identities of Trupanea spp. were confirmed by Dr. Elmo Hardy (University of Hawaii, Manoa). The identities of immature flies dissected from P. odorata flowerheads were confirmed by rearing them to adulthood. Voucher specimens of all fly species are stored in the Entomology Museum of the University of Hawaii at Manoa.

Data analysis. The relative abundance of flies associated with surveyed plants was calculated as a ratio of the number of emerging flies to the number of sampled flowerheads. The percent of P. odorata flowerheads infested by A. picturata was calculated as 100 ´ (number of flowerheads containing immature A. picturata/total number of flowerheads dissected). Comparisons among plant species and collection sites were made using Chi-square tests (Analytical Software, 1994). Seed production in infested and non-infested P. odorata flowerheads was compared using a t-test (Analytical Software, 1994).

Results and Discussion

A. picturata flies were reared from P. odorata, but not from flowerheads of any other plant species surveyed in this study (Table 3). As expected, flowerheads of E. mollis were infested only by T. obscuriventris, while flowerheads of S. oleraceous were infested only by E. sonchi. Among the native plants sampled, tephritid flies were reared only from the flowerheads of D. ciliolata (Trupanea sp. nr. cratericola) and D. laxa (T. dubautiae (Bryan)) (Table 3). Interestingly, E. sonchi was present in the samples of S. oleraceous flowerheads collected on the island of Kauai, but absent from all samples collected on the island of Hawaii. Previously, this species was recorded only from the island of Oahu (Hardy & Delfinado, 1980). Therefore, our findings indicate a geographical range expansion by E. sonchi, but this fly does not yet appear to spread to the island of Hawaii.

The ratio of the number of reared flies to the number of collected flowerheads varied widely among species (Chi-square test, df=4, χ²=32329.19, P<0.0001) (Table 3). Overall, A. picturata infested the lowest percentage of flowerheads, while T. obscuriventris infested the highest percentage (Table 3). The number of sampled flowerheads, location, and time of sampling differed depending on plant distribution and phenology. As a result, it was difficult to separate effects of insect, plant, and environmental factors, as well as their interactions. However, we detected significant differences among samples taken from the same plant species at different locations for P. odorata (df=9, χ;²=300.11, P<0.0001), for E. mollis (df=1, χ²=146.23.19, P<0.0001), and for S. oleraceous (df=1, χ²=4.27, P=0.0326) (Table 3). Levels of flowerhead infestation thus appear to be at least partially determined by microclimate or other environmental factors.

Table 3. Flowerhead infestation of selected adventive and endemic plants (family Asteraceae) surveyed on three Hawaiian islands in 1999-2000.

Plant species	Location	Time period	No. flowerheads collected	Fly species	No. adult flies	Fly : flowerhead ratio
Pluchea odorata	HAVO	April-Oct.1999	9721	Acinia picturata	98	1 : 99
	HAVO	March - April 2000	6013	Acinia picturata	25	1 : 241
	Pahala	April-Aug. 1999	8755	Acinia picturata	212	1 : 41
	Pahala	March - April 2000	13500	Acinia picturata	215	1 : 63
	Waiakea	March - April 2000	3343	Acinia picturata	89	1 : 38
	Kokee	March 1999	492	Acinia picturata	17	1 : 29
	Anini	Feb. - May 1999	5174	Acinia picturata	16	1 : 323
	KARC	Feb.-June 1999	7760	Acinia picturata	82	1 : 95
	Waimea Canyon	Feb.-March 1999	3361	Acinia picturata	7	1 : 480
	Old Pali Rd.	Feb.-March 2000	1768	Acinia picturata	56	1 : 32
	TOTAL for P. odorata		59886		817	1 : 73
Elephantopus mollis	Anini	Feb-June 1999	4463	Tetrauresta obscuriventris	4448	1 : 1
	KARC	Jan.-June 1999	9227	Tetrauresta obscuriventris	6670	1 : 1.4
	TOTAL for E. mollis		13690		11118	1 : 1.2
Sonchus oleraceous	Pahala	March-July 1999	2521	None	0	N/A
	Pahala	March - April 2000	4287	None	0	N/A
	Anini	March-June 1999	2456	Ensina sonchi	616	1 : 4
	KARC	April-June 1999	1078	Ensina sonchi	225	1 : 5
	TOTAL for S. oleraceous		3534		841	1 : 4
Bidens hawaiiensis	HAVO	May-Oct. 1999	431	None	0	N/A
Dubautia ciliolata	HAVO	April-Sept. 1999	15692	Trupanea sp. nr. cratericola	228	1 : 69
Dubautia laxa	Kaala	Oct. 1999	5215	Trupanea dubautia	2019	1 : 3
Dubautia plantaginia	Kaala	Oct. 1999	10961	None	0	N/A
Dubautia scabra	HAVO	April-Aug. 1999	7487	None	0	N/A
	HAVO	March - April 2000	5980	None	0	N/A
	TOTAL for D. scabra		13467

Dissections of P. odorata flowerheads revealed that only 13% of them contained immature stages of A. picturata. The observed infestation rate of dissected flowerheads was significantly higher than adult emergence rate from incubated flowerheads (Table 3), possibly due to the deleterious effects of experimental handling. None of the dissected flowerheads contained more than 1 insect, whether it was a larva or a pupa. Little is known about the oviposition behavior of A. picturata. However, females of a number of tephritid species have been reported to mark flowerheads with an oviposition-deterring pheromone when they deposit their eggs. In those species, conspecific females usually avoid laying eggs into the marked flowerheads, thus reducing intraspecific larval competition (Straw, 1989; Pittara & Katsoyanos, 1990; Lalonde & Roitberg, 1992). It is possible that similar behavior is responsible for the observed solitary distribution of immature A. picturata.

On average, flowerheads that contained A. picturata pupae produced 127.1 (SE=10.0) seeds, while uninfested flowerheads produced 299.0 (SE=13.4) seeds, indicating a 58% reduction due to larval feeding. In none of the infested flowerheads did larvae destroy all the seeds. The difference between infested and uninfested flowerheads was highly significant (t-test, df=62, t=10.25, P<0.0001). Visual inspection under a dissecting scope did not reveal any external differences between the undamaged seeds originating from infested and uninfested flowerheads. However, we made no attempt to compare seed germination rate or seedling viability.

Our results indicate that introducing A. picturata to Hawaii did not result in substantial impacts on populations of the target weed. While we did not detect any deleterious effects of this species on non-target vegetation, A. picturata populations also never reached the densities sufficient to suppress P. odorata. Even if developing larvae destroyed all the seeds in the flowerheads that they inhabited (and we have no reason to believe that this was the case), the maximum reduction in seed numbers barely reached 13%. If the 42% of seeds that in our study remained intact inside infested flowerheads were viable, then actual percent of destroyed seeds was more likely to be as low as 5 – 6%.

Very little is known about the biology and ecology of A. picturata, either within its original geographic range or in Hawaii, so it is hard to speculate about the reasons for such low fly densities. Fortunately, we did not uncover any evidence of host range expansion or effects on endemic species in the islands, although these endemics were not tested prior to the release of A. picturata. However, it appears that the A. picturata introduction failed to meet its original goal of suppressing P. odorata populations.

Acknowledgments

We thank Mike Klungness, Terri Moats, Ryan Nagata, Ed Acierto, and Malia Brown for technical assistance. We also thank the U.S. Department of the Interior and the Hawaii State Department of Land and Natural Resources for allowing us to conduct surveys on their lands. This work was supported in part by USDA-NRI Grant No. 98-351316-6982. This is Publication No. 4555 of the University of Hawaii, College of Tropical Agriculture and Human Resources Journal Series.

References

Analytical Software. (1996). Statistix for Windows, version 1.0. Analytical Software, Tallahassee, Florida.
ANDRES, L.A., DAVIS, C.J., HARRIS, P. & WAPSHERE, A.J. (1976) Biological control of weeds, in Theory and Practice of Biological Control (HUFFAKER, C.B. & MESSENGER, P.S., Eds.) Academic Press, New York, pp 481-500.
CENTER, T.D., FRANK, J.H. & DRAY, JR., F.A. (1995) Biological invasions: stemming the tide in Florida. Forida Entomologist 78, 45-55.
CHONG, M. (1962) Notes and exhibitions. Proceedings of the Hawaii Entomological Society 18, 25.
DAVIS, C J. (1961) Notes and exhibitions. Proceedings of the Hawaii Entomological Society 17, 315.
DAVIS, C.J. & KRAUSS, N.L.H. (1962) Recent developments in the biological control of weed pests in Hawaii. Proceedings of the Hawaii Entomological Society 18, 65-67.
DE BACH, P. & ROSEN, D. (1991) Biological Control by Natural Enemies. Cambridge University Press, Cambridge.
DUAN, J.J., PURCELL, M.F. & MESSING, R.H. (1996) Parasitoids of non-target tephritid flies in Hawaii: Implications for biological control of fruit fly pests. Entomophaga 41, 245-256.
GIAMBELLUCA, T.W., NULLET, M.A. & SCHROEDER, T.A. (1986) Rainfall Atlas of Hawaii. Honoulu Department of Land and Natural Resources, Divison of Water and Land Development.
HARDY, D.E. & DELFINADO M.D. (1980) Insects of Hawaii. vol. 13 - Diptera: Cyclorrhpha III. The University Press of Hawaii, Honolulu.
HARRIS, P. (1998) Evolution of classical weed biocontrol: meeting survival challenges. Bulletin of the Entomological Society of Canada 30, 134-143.
HOPPER, K.R. (1998) Assessing and improving the safety of introductions for biological control. Phytoprotection 79(S), 84-93.
HOWARTH, F.G. (1991) Environmental impacts of classical biological control. Annual Review of Entomology 36, 485-509.
LALONDE, R.G. & ROITBERG, B.D. (1992) Host selective behavior of a thistle-feeding fly: choices and consequences. Oecologia 90, 534-539.
LOUDA, S.M., KENDALL, D., CONNOR, J. & SIMBERLOFF, D. (1997) Ecological effects of an insect introduced for the biological control of weeds. Science 277, 1088-1090.
MCEVOY, P.B. (1996) Host specificity and biological pest control. Bioscience 46, 401-405.
MUELLER-DOMBOIS, D. & FOSBERG, F.R. (1974) Vegetation map of Hawaii Volcano National Park. Technical Report No. 4, Coop. Natn’l Park Resources Studies Unit.
PITTARA, I.S. & KATSOYANOS, B.I. (1990) Evidence for a host-marking pheromone in Chaetorellia australis. Entomologia Experementalis et Applicata 54, 287-295.
RIPPERTON, J.C. & HOSAKA, E.Y. (1942) Vegetation zones of Hawaii. Hawaii Agricultural Experiment Station Bulletin No. 89, Honolulu, HI.
SIMBERLOFF, D. & STILING, P. (1996) How risky is biological control? Ecology 77, 1965-1974.
SMITH, C.W. (1985) Impact of alien plants on Hawaii’s native biota, in Hawaii’s Terrestrial Ecosystems: Preservation and Management (STONE, C.P. & SCOTT, J.M., Eds.) Coop. Natl. Park. Res. Study Unit, University of Hawai`i, Honolulu, HI. pp 180-250.
SOHMER, S.H. & GUSTAFSON, R. (1987) Plants and Flowers of Hawaii. University of Hawaii Press, Honolulu, HI.
STRAW, N.A. (1989) Evidence for an oviposition-deterring pheromone in Tephritis bardanae (Schrank) (Diptera: Tephritidae). Oecologia 78, 121-130.
VAN DRIESCHE, R.G. & BELLOWS, JR., T.S. (1996) Biological Control. Chapman and Hall, New York.
WAGNER, W.L., HERBST, D.R. &. SOHMER, S.H. (1999) Manual of the Flowering Plants of Hawaii. University of Hawaii Press and Bishop Museumn Press, Honolulu.
WAPSHERE, A.J. (1974). A strategy for evaluating the safety of organisms for biological weed control. Annals of Applied Biology 77, 201-211.
WAPSHERE, A.J. (1989) A testing sequence for reducing the rejection of potential biological control agents for weeds. Annals of Applied Biology 114, 515-526.
WHISTLER, W.A. (1995) Wayside Plants of the Islands: A Guide to the Lowland Flora of the Pacific Islands Including Hawaii, Samoa, Tonga, Tahiti, Fiji, Guam, Belau. Isle Botanica, Honolulu.
WILLIAMSON, M. & FITTER, A. (1996) The varying success of invaders. Ecology 77, 1661-1666.