Unsuccessful introduced biocontrol agents can act as pollinators of invasive weeds: Bitou Bush (Chrysanthemoides monilifera ssp. rotundata) as an example

The extent of self‐compatibility and reliance on pollinators for seed set are critical determinants of reproductive success in invasive plant species. Seed herbivores are commonly used as biocontrol agents but may also act as flower visitors, potentially resulting in pollination. However, such contrasting or potentially counterproductive interaction effects are rarely considered or evaluated for biological control programs. We investigated the breeding system and pollinators of Bitou Bush (Chrysanthemoides monilifera ssp. rotundata), an invasive species in Australia that has been the subject of biocontrol programs since 1987. We found the species to be obligate outcrossing in all six populations tested. From 150 video hours, we found 21 species of potential pollinators, including Mesoclanis polana, the Bitou Seedfly, native to South Africa and released in Australia as a biocontrol agent in 1996. Mesoclanis polana transferred pollen to stigmas and was the most common pollinator (52% of pollinator visits), followed by the syrphid fly Simosyrphus grandicornis (9%) and introduced honeybee, Apis mellifera (6.5%). Fruit‐to‐flower ratios ranged from 0.12 to 0.45 and were highest in the population with the greatest proportion of Mesoclanis polana visits. In an experimental trial, outside the naturalized range, the native bee Homalictus sphecodoides and the native syrphid Melangyna viridiceps were the primary pollinators, and fruit‐to‐flower ratios were 0.35, indicating that Bitou Bush would have ready pollinators if its range expanded inland. Synthesis. Invasive Bitou Bush requires pollinators, and this is effected by a range of generalist pollinators in eastern Australia including the Bitou Seedfly, introduced as a biocontrol agent, and the major pollinator detected in this study. Fruit‐to‐flower ratios were highest when the Bitou Seedfly was in high abundance. This study underscores the importance of evaluating the pollination biology of invasive species in their native ranges and prior to the introduction of biocontrol agents.     

Impact of the biological control agent Mesoclanis polana (Tephritidae) on bitou bush (Chrysanthemoides monilifera subsp. rotundata) in eastern Australia

The seed fly Mesoclanis polana (Diptera: Tephritidae) was released in Australia in 1996. Its impact on seed production of bitou bush (Chrysanthemoides monilifera subsp. rotundata) was monitored at eight sites along the New South Wales coast from 1996 to 2004. Peak flowerhead production occurred in autumn (March to May); therefore, samples collected in May of each year were used to compare abundance and impact of M. polana across sites and between years. Latitude had a significant effect on abundance and impact of M. polana. By May 2004, 99.6% of flowerheads at the five most northern sites contained at least one egg, while 64% of flowerheads from the two most southern sites contained at least one egg. In May 2004, mean numbers of M. polana eggs per flowerhead were between 13 and 17 at four of the six northern sites and below two at the two southern sites. In May 2004, average seed destruction by M. polana was 58% at the four most northern sites and 11% at the two most southern sites. The highest level of seed destruction recorded was 86% at Kingscliff in May 2003. Percentage seed destruction in May provided a reasonable estimate of seed destruction for the whole year. Parasitoids were found throughout the range of M. polana in Australia. Their attack rate on M. polana was less than 10% at all sites, except at the two most northern sites where parasitism of up to 27% was recorded. Parasitism results are compared with predictions made following an earlier study. The release of Mesoclanis magnipalpis, a species suited to cooler environments, is recommended for the southern range of C. monilifera subsp. rotundata.     

Soil‐mediated effects on germination and seedling growth of coastal wattle (Acacia sophorae) by the environmental weed,bitou bush (Chrysanthemoides monilifera ssp. rotundata)

Bitou bush (Chrysanthemoides monilifera ssp. rotundata; Asteraceae) is a major woody weed that competes with the native legume Acacia sophorae in coastal ecosystems of eastern Australia. Three glasshouse experiments examined whether litter or soil from beneath bitou bush or Acacia plants could influence seed germination and seedling growth of A. sophorae. The presence of litter decreased seed germinability and this effect was greater for bitou bush litter than for Acacia litter. Shoot growth was increased by the addition of Rhizobium after 40 days, irrespective of soil type. After 78 days, shoot and root biomass were significantly lower for seedlings grown in bitou bush soil than for those grown in Acacia soil. There was a non‐significant trend towards a lower median population of Rhizobium in the soil beneath bitou bush than in that beneath Acacia. The results demonstrated a slight effect of bitou bush on the growth of A. sophorae, which could, however, be overshadowed by the judicious use of herbicides or fire for weed control and revegetation.     

Integrating management techniques to restore subtropical forests invaded by Hedychium coronarium J. Köenig (Zingiberaceae) in a biodiversity hotspot

The restoration of areas invaded by non‐native plants is challenging as invasive plants may affect both biotic and abiotic components of ecosystems, leading to impacts that constrain recolonization by native species after invaders are eliminated. In such a scenario, restoration techniques as topsoil transposition might accelerate colonization by native species in forests. Hedychium coronarium J. Koenig (Zingiberaceae) is a Himalayan herbaceous rhizomatous plant recognized as invasive in several countries. This study aimed to experimentally evaluate the response of plant assemblages to topsoil transposition on a site invaded by H. coronarium after chemical control. Four treatments were applied: chemical control integrated with topsoil transposition, chemical control of H. coronarium alone, topsoil transposition alone, and no intervention (control). Plots were evaluated prior to the application of treatments and then monthly for 11 months after treatments. Parameters were measured for H. coronarium (number of ramets, ramet height, and cover) and other species (species richness, abundance, and cover). Plots treated with chemical control (regardless of topsoil transposition) were similar in terms of all parameters measured and species composition, with dominance of herbs and shrubs. Plots managed solely with topsoil transposition had lower species richness, abundance, and cover, but more diverse life‐forms, being equally rich in climbers, trees, and herbs. Chemical control was effective to control invasion by H. coronarium and increase species richness and abundance on the managed site. Topsoil transposition promoted colonization by species that might accelerate restoration.     

Ecological impacts of invasive African olive (Olea europaea ssp. cuspidata) in Cumberland Plain Woodland,Sydney, Australia

African olive (Olea europaea ssp. cuspidata) is a small evergreen tree which has become highly invasive at a landscape scale in the western Sydney and Hunter Valley regions of New South Wales, Australia. African olive invasion results in the formation of a dense and permanent mid‐canopy in grassy woodland vegetation. We investigated the relationship between African olive and native species establishment, abundance and diversity, using field surveys and a manipulative shading experiment. There were 78% fewer native species beneath African olive canopy in the field compared to uninvaded woodland sites. The shading experiment showed that simulated dense African olive shade levels produced the lowest dry weight for the three native species studied, with simulated canopy edge light providing optimal conditions for the native shrub Bursaria spinosa and African olive. Dense African olive shade levels produced the highest mortality rate for native species; however, African olive was able to maintain an 88% survival rate under dense canopy shade. This study confirms the adaptability of African olive and its capacity to decrease native plant diversity and substantially modify native vegetation at the community level.     

The ecological barriers to the recovery of bridal creeper (Asparagus asparagoides (L.) Druce) infested sites: Impacts on vegetation and the potential increase in other exotic species

Abstract To protect native biodiversity from environmental weeds, the impacts that these weeds cause need to be known before weed control commences. Asparagus asparagoides (L.) Druce (bridal creeper) (Asparagaceae) is a serious environmental weed and has been selected for biological control in Australia. To predict the responses of plant communities to the control of bridal creeper, a prerelease baseline of the impacts of bridal creeper on native plant communities was undertaken. Plant assemblages in areas invaded by bridal creeper were compared with reference areas that contained little or no bridal creeper. Areas invaded by bridal creeper contained 52% fewer native plant species when compared with nearby reference areas. However, there was no difference in the number of other exotic plant species between areas. Similar trends were found for the germinable seed bank. Although a greater number of exotic species were present in the seed bank compared with the vegetation surveys, there was still no difference between areas with and without bridal creeper. In a glasshouse trial, exotic species germinated more frequently compared with native species. This could indicate that as bridal creeper density decreases following control, exotic species have an advantage over native species when colonizing areas left vacant by bridal creeper. Second, as bridal creeper areas contained reduced native species richness and cover, they may be susceptible to further weed invasion after bridal creeper is removed. Therefore, simply reducing the presence of bridal creeper may not guarantee successful restoration of invaded areas and additional restoration efforts will be needed to ensure the ultimate goal of protecting native biodiversity is reached.     

Integrating flood depth and plant resistance with chlorantraniliprole seed treatments for management of rice water weevil,Lissorhoptrus oryzophilus (Coleoptera: Curculionidae)

Chlorantraniliprole seed treatments in rice provide effective suppression of rice water weevil populations in the United States; however, heavy reliance on prophylactic insecticide treatments as a sole strategy could destabilize management programs for this insect. The present research evaluated the compatibility of seed treatments with two other potential management tactics—plant resistance and shallow flooding–by conducting two split‐plot experiments in 2009 and 2011. In both experiments, no substantial antagonism was found among the 3 different tactics. Statistical interactions in these experiments arose from the strong and persistent effects of chlorantraniliprole on larval densities rather than incompatibility of tactics. In 2009, weevil densities differed among varieties and were significantly lower on the cultivar “Jefferson.” In 2011, weevil densities were reduced significantly in shallow‐flooded plots compared to deep‐flooded plots. Significant reductions in weevil numbers by chlorantraniliprole seed treatments, even at application rates 5 fold lower than commercially recommended rates, demonstrated the potential to reduce application rates of this highly potent larvicide. These latter results suggest that future studies on the relationship between chlorantraniliprole seed treatment rate and weevil fitness are warranted.     

Host range validation,molecular identification and release and establishment of a Chinese biotype of the Asian leaf beetle Lilioceris cheni (Coleoptera: Chrysomelidae: Criocerinae) for control of Dioscorea bulbifera L. in the southern United States

Dioscorea bulbifera, an Asian vine, is invasive in the southeastern USA. It rarely flowers but propagates from potato-like bulbils formed in leaf axils, which persist into the subsequent growing season. Lilioceris cheni Gressitt and Kimoto, a foliage-feeding beetle (Coleoptera: Chrysomelidae: Criocerinae) from Nepal, had been tested, proven to be a specialist and approved for release as a biological control agent. Regulatory delays, however, resulted in the demise of quarantine-held colonies, and acquisition of new Nepalese stock proved untenable. Searches then undertaken in southern China resulted in the collection of over 300 similar beetles. Two Chinese Lilioceris species were identified: one confirmed to be L. cheni and the other identified as Lilioceris egena (Weise). Mitochondrial analysis revealed an exact DNA match between some Chinese and one of the two Nepalese c oxidase subunit I haplotypes and all Chinese L. cheni haplotypes clustered as a single species but the comingling of the two species aroused concerns over possible hybridisation. These concerns were allayed by nuclear D2 analysis showing the absence of dual parental sequences. Nonetheless, diligence was exercised to ensure that the Chinese strains were safe to release. Abridged host testing using critical test species verified specificity. Caged releases during autumn 2011 documented the ability of adult beetles to overwinter in south Florida despite a prolonged lack of foliage. Open releases the following year produced vigorous populations that caused extensive defoliation. Preliminary observations indicate that L. cheni now contributes to the control of D. bulbifera and the bulbil-feeding L. egena should complement these effects if its host range proves appropriate.     

Biological Control of Hedge Bindweed (Calystegia sepium) with Stagonospora convolvuli Strain LA39 in Combination with Competition from Red Clover (Trifolium pratense)

In a maize cropping system where a living green cover suppresses many weeds, Calystegia sepium is able to escape control. In this paper we report the potential for biological control of C. sepium by using the bindweed pathogen Stagonospora convolvuli strain LA39 as a mycoherbicide in combination with competition by the green cover plant Trifolium pratense. In a greenhouse experiment, competition from shoots of T. pratense caused a strong reduction of the biomass of C. sepium, and combined competition from shoots and roots had the same effect. In a second, factorial greenhouse experiment, competition by T. pratense again reduced C. sepium biomass. However, S. convolvuli did not influence the number of leaves or the biomass of C. sepium in the greenhouse even though severe necrosis was observed on inoculated bindweed leaves. In contrast, in a 2-year field study, S. convolvuli caused severe disease and a strong reduction of C. sepium ground coverage in maize. Underseeding with T. pratense had no effect on disease severity, but T. pratense reduced ground coverage by C. sepium at one of eight samplings in the first year. In conclusion, S. convolvuli is useful in the field and, as shown in the greenhouse, a competitive green cover might improve biological control of C. sepium.     

Landscape-scale detection and mapping of invasive African Olive (Olea europaea L. ssp. cuspidata Wall ex G. Don Ciferri) in SW Sydney,Australia using satellite remote sensing

Question: Is satellite imagery an effective tool for mapping and examining the distribution of the invasive species Olea europaea L. ssp. cuspidata at a regional landscape scale? Location: Southwest Sydney, Australia. Methods: Remote sensing software was used to classify pixels of Olea europaea L. ssp. cuspidata (African Olive) and major vegetation types from satellite imagery, using a “supervised classification” technique across a 721 km² study area in the Cumberland Plain region of western Sydney. A map of African Olive distribution was produced from the image analysis and checked for accuracy at 337 random locations using ground observation and comparison with existing vegetation maps. The African Olive distribution data were then used in a GIS analysis with additional spatial datasets to investigate the relationship between the distribution of African Olive and environmental factors, and to quantify the conservation threat to endangered native vegetation. Results: A total area of 1907 ha of dense African Olive infestation was identified, with an omission error of 7.5% and a commission error of 5.4%. African Olive was found to occur on the steepest slopes (mean slope 14.3°) of the vegetation classes examined, with aspect analysis identifying a high prevalence on south- and southwest-facing slopes. The analysis also quantified the level of African Olive infestation in endangered ecological communities, with Western Sydney Dry Rainforest (25% affected) and Moist Shale Woodland (28% affected) identified as most vulnerable to African Olive invasion. Conclusion: The distribution of African Olive can be efficiently mapped at a landscape scale. This technique, used in association with additional spatial datasets, identified African Olive as a significant environmental weed in SW Sydney, occupying a greater area than previously recognised and threatening several endangered native vegetation communities.     

Environmental conditions and phosphorus removal in Florida lakes and wetlands inhabited by Hydrilla verticillata (Royle): implications for invasive species management

Hydrilla verticillata is considered the most problematic aquatic plant in the United States. In south Florida, Hydrilla dominance has also been documented in treatment wetlands. This paper characterizes (1) environmental conditions which favor Hydrilla growth and (2) understand its nutrient removal capability. Despite its occurrence over a wide range of environmental conditions, Hydrilla abundance increased with increasing pH, alkalinity, total P and total N, and decreased with water depth in selected Florida lakes. No relationship was found between color, Secchi depth and Hydrilla abundance. In several Hydrilla-dominated lakes, mean total P concentration (126 μg/l) at inflow was reduced to 106 μg/l at outflow. The maximum inflow total P concentration in a lake with positive nutrient reduction was 148 μg/l. Total P removal efficiency by Hydrilla-dominated lakes and wetlands was comparable to or higher than systems dominated by emergent and other submerged plants. Mean total P settling rates for lakes and a constructed wetland dominated by Hydrilla were estimated at 19 and 34 m/year, respectively, which were higher than or comparable to similar systems dominated by other aquatic plants. Results from this study suggest that reduction of Hydrilla from constructed wetlands will not likely improve nutrient removal performance.     

Invasive species can't cover their tracks: using microsatellites to assist management of starling (Sturnus vulgaris) populations in Western Australia

Invasive species are known to cause environmental and economic damage, requiring management by control agencies worldwide. These species often become well established in new environments long before their detection, resulting in a lack of knowledge regarding their history and dynamics. When new invasions are discovered, information regarding the source and pathway of the invasion, and the degree of connectivity with other populations can greatly benefit management strategies. Here we use invasive common starling ( Sturnus vulgaris ) populations from Australia to demonstrate that genetic techniques can provide this information to aid management, even when applied to highly vagile species over continental scales. Analysis of data from 11 microsatellites in 662 individuals sampled at 17 localities across their introduced range in Australia revealed four populations. One population consisted of all sampling sites from the expansion front in Western Australia, where control efforts are focused. Despite evidence of genetic exchange over both contemporary and historical timescales, gene flow is low between this population and all three more easterly populations. This suggests that localized control of starlings on the expansion front may be an achievable goal and the long-standing practice of targeting select proximal eastern source populations may be ineffective on its own. However, even with low levels of gene flow, successful control of starlings on the expansion front will require vigilance, and genetic monitoring of this population can provide essential information to managers. The techniques used here are broadly applicable to invasive populations worldwide.     

Response ofHelicoverpa spp. (Lep.: Noctuidae) and their natural enemies to petroleum spray oil in cotton in Australia

Refined petroleum products have been used since last century for the control of phytophagous arthropod pests. The response ofHelicoverpa spp. and its natural enemies to petroleum oil sprays on cotton was investigated under laboratory choice/no-choice and small plot field conditions at the Australian Cotton Research Institute at Narrabri in New South Wales. In oviposition choice and no-choice tests, petroleum oil sprays suppressed oviposition inHelicoverpa punctigera (Wallengren) but notH. armigera (Hubner) (Lepidoptera: Noctuidae).H. punctigera females laid about seven times as many eggs on the control (water sprayed) cotton plants as on either petroleum oil spray alone or oil mixed with Kelgum. HoweverH. armigera females laid the same number of eggs on both treated and control plants. In field trials, the number of eggs per metre per sample date found on the plots treated with petroleum oil spray was 3.83 compared with 6.29 and 7.82 eggs respectively on plots treated with conventional insecticide sprays and the unsprayed plots. The number of larvae found on plots treated with petroleum oil sprays was 2.9 times higher than the conventional insecticide sprayed plot, however, this was 1.6 times fewer than the unsprayed plot. The cotton yields from plots treated with petroleum oil sprays was 3.70 bales/ha compared with 7.32 and 2.69 bales respectively from the conventional insecticide treated and the unsprayed plots. Petroleum oil sprays had little or no effect on predatory beetles, bugs, lacewings and spiders.

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Résumé Des produits de pétrole raffiné ont été utilisés depuis le siècle dernier pour lutter contre les arthropodes ravageurs phytophages. La réponse deHelicoverpa spp. et des ses ennemis naturels à des pulvérisations d'huile de pétrole sur cotonnier a été étudiée en laboratoire dans des expériences de choix et d'absence de choix ainsi que dans de petites parcelles en plein champ à l'Institut de Recherche australien sur le coton (Narrabri, Nouvelles Galles du Sud). Dans des expériences de choix/non choix concernant l'oviposition, les pulvérisations d'huile de pétrole empêchaient la ponte d'Helicoverpa punctigera (Wallengren) mais pas deH. armigera (Hubner) (Lep.: Noctuidae). Les femelles deH. punctigera pondent environ 7 fois plus d'oeufs sur les plants de coton témoins (pulvérisés avec de l'eau) que sur les plants traités à l'huile de pétrole seule ou en mélange avec du Kelgum. Par contre,H. armigera pond le même nombre d'oeufs sur les plants traités ou témoins. Dans les essais en plein champ, les parcelles pulvérisées avec de l'huile de pétrole comportent significativement moins deHelicoverpa et présentent une récolte plus importante que celles non traitées; cependant le nombre de chenilles est plus élevé et la récolte moindre que dans les parcelles traitées avec des insecticides. Les pulvérisations d'huile de pétrole ont un effet faible ou nul sur la faune auxiliaire.

     

Adaptive major histocompatibility complex (MHC) and neutral genetic variation in two native Baltic Sea fishes (perch Perca fluviatilis and zander Sander lucioperca) with comparisons to an introduced and disease susceptible population in Australia (P. fluviatilis): assessing the risk of disease epidemics

This study assessed the major histocompatibility complex (MHC) and neutral genetic variation and structure in two percid species, perch Perca fluviatilis and zander Sander lucioperca, in a unique brackish ecosystem, the Baltic Sea. In addition, to assess the importance of MHC diversity to disease susceptibility in these populations, comparisons were made to an introduced, disease susceptible, P. fluviatilis population in Australia. Eighty‐three MHC class II B exon 2 variants were amplified: 71 variants from 92 P. fluviatilis samples, and 12 variants from 82 S. lucioperca samples. Microsatellite and MHC data revealed strong spatial genetic structure in S. lucioperca, but not P. fluviatilis, across the Baltic Sea. Both microsatellite and MHC data showed higher levels of genetic diversity in P. fluviatilis from the Baltic Sea compared to Australia, which may have facilitated the spread of an endemic virus, EHNV in the Australian population. The relatively high levels of genetic variation in the Baltic Sea populations, together with spatial genetic structure, however, suggest that there currently seems to be little risk of disease epidemics in this system. To ensure this remains the case in the face of ongoing environmental changes, fisheries and habitat disturbance, the conservation of local‐scale genetic variation is recommended.     

Comparison of different methods to construct a core germplasm collection in woody perennial species with simple sequence repeat markers. A case study in cherimoya (Annona cherimola, Annonaceae), an underutilised subtropical fruit tree species

Although molecular markers are becoming the tool of choice to develop core collections in plants, the examples of their use in woody perennial species are very scarce. In this work, we used simple sequence repeat (SSR) marker data to develop a core collection in an underutilised subtropical fruit tree species, cherimoya ( Annona cherimola , Annonaceae), from an initial collection of 279 genotypes from different countries. We compared six alternative allocation methods to construct the core collection, four not based upon the similarity dendrogram [random sampling, maximisation strategy (M strategy) and simulated annealing algorithm maximising both genetic diversity and number of SSR alleles] and two based on dendrogram data (logarithmic strategy and stepwise clustering). The diversity maintained in each subset was compared with that present in the entire collection. The results obtained indicate that the use of SSRs together with the M strategy is the most efficient method to develop a core collection in cherimoya. In the best subset, with 40 accessions, all the SSR alleles present in the whole collection were recovered and no significant differences in frequency distribution of alleles for any of the loci studied or in variability parameters ( H _O, H _E) were recorded between the core and the whole collection.     

Historical tests of the toxicity of pesticides to Typhlodromus pyri (Acari: Phytoseiidae) and their relevance to current pest management in New Zealand apple orchards 1. Laboratory tests with eggs and larvae

A two-part review is presented relating historical tests of the toxicity of pesticides to Typhlodromus pyri and their relevance to modern pest management in New Zealand pome-fruit orchards. Over the past thirty years, the initial need for T. pyri resistance to broad-spectrum pesticides has substantially declined as a growing array of new selective chemicals have come into use. In Part 1, a laboratory bioassay is described for determining the toxicity of pesticides to the eggs and larvae of an organophosphate (OP)-resistant strain of Typhlodromus pyri from New Zealand. Apple leaves bearing T. pyri and its prey Panonychus ulmi were collected from the field. Leaf discs with known numbers of eggs (no active stages) of T. pyri and prey were cut from the leaves and sprayed with selected pesticides at recommended field rates to simulate field application. The survival of eggs, and the larvae which hatched from them, were recorded for seven days. Thirteen acaricides, 16 fungicides and 15 insecticides were evaluated. Toxic chemicals were aminocarb, amitraz, benomyl, binapacryl, chlordimeform, ethion, omethoate, oxamyl, permethrin, pirimiphos-methyl and triazophos. Slight and variable toxicity was caused by azinphos-methyl, chlorpyrifos, dinocap, mancozeb + dinocap, metiram + nitrothal-isopropyl, and sulphur. No toxicity was detected with the other 24 pesticides. A comparison of the test results with those from field trials in New Zealand showed good agreement, except that the laboratory tests failed to detect the known field toxicity of dithiocarbamate fungicides and the insecticide vamidothion. Most of the chemicals tested are no longer used in commercial pome-fruit orchards in New Zealand, all of which now practise integrated (IFP) or organic (OFP) fruit production based on selective pest management methods. The tested pesticides of continuing importance are identified, and a summary is presented of the international literature describing the impact on T. pyri of the current pesticides used in New Zealand IFP and OFP. The changes in pesticide use in New Zealand are paralleled by similar changes in most pome-fruit growing areas of the world.     

The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes. Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-γ and tumour necrosis factor-β, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population and that these cells were the major source of interferon-γ. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen-specific Th1-like cells, PSA-2 might be considered a vaccine candidate for human leishmaniasis.