Establishment of Citrostichus phyllocnistoides (Hymenoptera: Eulophidae) as a biological control agent for the citrus leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae) in Spain

BIOLOGICAL CONTROLÐPARASITOIDS AND PREDATORS

Recruitment of Native Parasitoid Species by the Invading Leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae) on Citrus in Spain R. VERCHER, J. COSTA-COMELLES, C. MARZAL

AND

F. GARCI´A-MARI´1

Institut Agroforestal Mediterrani, Universitat Polite` cnica de Vale` ncia, Camõ´ de Vera 14, 46022 Vale` ncia, Spain

Environ. Entomol. 34(5): 1129Ð1138 (2005)

ABSTRACT The parasitoid assemblage associated with the citrus leafminer Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) was studied in citrus orchards in eastern Spain over a 7-yr period (1995Ð2001) after the leafminerÕs introduction in 1994. In total, 11,587 adult native parasitoids were collected. To evaluate parasitism, 93,846 live immature stages of the citrus leafminer were observed, of which 21,460 (22.9%) were found to be parasitized. The parasitoid complex recruited around P. citrella was typical for invader hosts: lower species richness, generalized habits, idiobiont strategy, and low to moderate rates of parasitism. Two of the 10 species reared from the citrus leafminer, Pnigalio sp. and Cirrospilus brevis Zhu, LaSalle and Huang, accounted for ⬎90% of the parasitoids. Native parasitoids moved onto the invading host rapidly, except for C. brevis, which required 3 yr to become common and widespread. In other leafminer species from plants in the vicinity of citrus orchards, the proportion of P. citrella parasitoids was higher in woody (69.7%) than in herbaceous plants (22.2%). The high population levels reached by the new pest, associated with a negative density dependence response of the parasitoids at these high population levels, suggest that the native parasitoid assemblage exerted only a limited role in regulating the population of the new host. KEY WORDS Phyllocnistis citrella, Cirrospilus brevis, Pnigalio, parasitism, citrus

THE CITRUS LEAFMINER, Phyllocnistis citrella Stainton, is a microlepidopteran pest of citrus native to eastern and southern Asia. It invaded most citrus-growing regions of the Americas and the Mediterranean basin in the early 1990s. In the Mediterranean region, it reached high population levels and caused extensive damage to new ßushes, particularly in young trees, nurseries, and overgraftings (Garcõ´a-Marõ´ et al. 2002). The alien pest was initially controlled using insecticides, but it soon became evident that chemical control was a costly and short-term solution (Knapp et al. 1995, Argov and Ro¨ ssler 1996). The development of a biological control program seemed to be the best option to control this exotic insect, as has been done for other invasive pests with similar characteristics (Greathead and Greathead 1992). Classical biological control efforts aimed at the introduction of host-speciÞc insects from within the native range of the citrus leafminer were implemented in several places such as Australia, the United States, and various countries of the Mediterranean area (Hoy and Nguyen 1995, Neale et al. 1995, Argov and Ro¨ ssler 1996, FAO 1996, Vercher 2000, Siscaro et al. 2003). The parasitoids most commonly introduced were Agenias1 Corresponding author: Entomologia, Departament dÕEcosistemes, ETSEA, Universitat Polite` cnica de Vale` ncia, Camõ´ de Vera 14, 46022 Vale` ncia, Spain (e-mail: [email protected]).

pis citricola (Logvinosvskaya), Cirrospilus ingenuus (Gahan), Quadrastichus citrella Reina and La Salle, Semielacher petiolatus (Girault), and Citrostichus phyllocnistoides (Narayanan). In Spain, the citrus leafminer was Þrst detected in 1993, expanded in 1994, and was fully established in all the citrus areas by 1995. In 1996, a classical biological control targeting the citrus leafminer was initiated. Five species of exotic parasitoids were released in the following years (Vercher et al. 2000), but until 1999, only native species were found parasitizing the new host. From 2000 onward, the exotic parasitoid C. phyllocnistoides, Þrst released in 1998, became established and dispersed, reaching high parasitism rates and lowering citrus leafminer population (Garcõ´a-Marõ´ et al. 2003). Simultaneously, a long-term study was developed in the Spanish Mediterranean area to determine the structure of the community of native parasitoids associated with the new pest and to understand more about the factors determining host breadth. In this paper, we present and analyze the development of the parasitoid assemblage associated with the invading pest over a 7-yr period. Recruitment of native parasitoids by P. citrella was followed from the beginning of the pest establishment, starting in 1995 when the citrus leafminer had spread into all citrus growing areas of eastern Spain, and ending in 2001, when the exotic parasitoid C. phyllocnistoides became estab-

0046-225X/05/1129Ð1138$04.00/0 䉷 2005 Entomological Society of America

1130 Table 1.

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Number of samples made in citrus orchards in the eastern Spain region between 1995 and 2001

Geographic area

Number of localities

Number of orchards

Ribera Baixa Ribera Alta Valldigna Horta Nord Camp de Morvedre Camp de Turia Total

4 6 4 4 2 1 21

21 28 16 25 12 3 105

Number of samples 1995

1996

1997

1998

1999

2000

2001

12 6 8 6 1

86 23 51 51 46 1 258

57 36 56 70 25 8 252

57 49 51 42 15 27 241

33 52 5 33 31 34 188

40 40

37 38

40 40 40 200

38 38 38 189

33

lished in all the citrus producing area, displacing and disrupting the assemblage of native parasitoids on citrus leafminer. The temporal dynamics of parasitoids, some hostÐparasitoid interactions, and other aspects of the life history of the parasitoids also are reported. To better understand parasitoid recruitment into the orchards, the study included sampling of the parasitoid assemblages of the leafmining guild outside of the orchards to ascertain the pool of available parasitoid species and perhaps determine the sources of parasitoids colonizing this exotic pest. Materials and Methods Sampling Sites. The citrus region of eastern Spain forms a continuum of cultivated citrus orchards along the Mediterranean coast, ⬇200 km from north to south and 20 Ð50 km wide, with a crop surface of ⬃180,000 ha. Valencia is situated approximately in the center of this area. Samples were taken from citrus orchards selected within an area of 60 km around Valencia, distributed in six different geographic areas. From 1995 to 2001, 1,361 samples were made (Table 1). In 1995, 33 samples were collected between September and November. Between 1996 and 1999, 188 Ð258 samples were collected annually whenever citrus leafminer was present in young shoots (usually May to November). Samples came from a total of 105 different citrus orchards, each sampled between one and six times a year. In 2000 and 2001, 10 orchards were sampled every 2 wk throughout the citrus growing season, representing a total of 200 samples in 2000 and 189 samples in 2001 (Table 1). The samples collected each year were from a similar period of time, taken regularly along this period and in similar geographic areas. The orchards sampled were 1- to 2-ha commercial plantations that underwent the cultural practices usually applied in the area but were not sprayed to control P. citrella throughout the sampling period. Usually, one or two chemical treatments were applied annually in the orchards to control other pests, generally in the middle of the sampling period (August or September). Approximately 30% of the samples were collected from orchards not treated at least during 1 yr. These orchards were either undergoing organic farming or owners agreed not to apply chemical sprays. Level of Parasitism and Parasitoid Assemblage Associated with P. citrella. Samples from citrus orchards consisted of 100 Ð150 developed new shoots contain-

ing different life stages of the citrus leafminer. Shoots were randomly collected, placed in a plastic bag, and transported to the laboratory. The percentage of parasitism was estimated from 1995 through 1999. Fifty living leafminers of each susceptible life stage (second, third, and fourth larval instars and pupae) were observed under a stereomicroscope to detect the presence of parasitoids. Cases of hyperparasitism or superparasitism also were recorded when observed. Percentages of parasitism for each susceptible stage of P. citrella were summed and divided by the number of susceptible stages to give an estimate of the global parasitism rate. Immature parasitoids found in the mines were isolated in glass vials, placed in climate controlled chambers (25 ⫾ 2⬚C, 50 Ð 60% RH, and LD 16:8 h), and allowed to develop to adult stage for identiÞcation. The preference of each parasitoid species for different stages of P. citrella was determined by identifying the parasitoids that emerged from each one of the stages of P. citrella parasitized. All native parasitoids detected on P. citrella were idiobiont species (Vercher 2000), as is usual in leafminer parasitoids (Hawkins et al. 1992, Cornell and Hawkins 1993, Askew 1994), so we could assume that the stage in which we found the parasitoids was the stage attacked initially by ovipositing females. Altogether, 5,508 collected P. citrella yielded adult parasitoids. The rest of the sample was enclosed in 3-liter glass jars covered internally with Þlter paper, closed with a mesh cloth, and placed into the climatic chamber. After 30 d, jars were opened, and the debris and desiccated plant material was sieved to collect the emerged adult parasitoids, which were counted and identiÞed to species. All shoots of samples collected in 2000 and 2001 were held in the 3-liter glass jars until parasitoids emerged, after which they were counted and identiÞed. The percentage of parasitism inßicted by native parasitoids was thus not estimated in this period. Population Density of P. citrella. There is a close relationship between the percentage of damaged leaf area and the number of mines per leaf of P. citrella (Pen˜ a et al. 2000). The abundance of the citrus leafminer was therefore determined by estimating the percentage of damaged leaf area. The number of mines per leaf was calculated using the formula y ⫽ x2/1,220, x being the percentage of foliar surface lost and y the number of mines per leaf. This formula was obtained from studies carried out in the same area

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(Vercher 2000). The damage was estimated on 514 samples taken from 1996 to 1999 (between May and November) in the orchards most frequently sampled. Fifteen new shoots were randomly collected per sample. Shoots with fully expanded leaves were purposely selected for this evaluation to guarantee that leafminer damage had been completed. In the laboratory, the percentage of leaf surface damaged by mining was estimated by visual observation, qualifying each leaf from 0 to 10 as the leaf area damaged ranged from 0 to 100% (Schaffer et al. 1997). To study the relationship between citrus leafminer population density and parasitism levels, samples were grouped according to the average number of mines developed per leaf, from zero to one, from more than one to two, and so on up to six mines per leaf, the maximum average number of mines found per sample. Abundance of Parasitoids in Other Leafminers. Herbaceous and woody plants attacked by leafminers and located in the proximity of the target citrus orchards were sampled from 1996 to 1998 to understand the host complex used by native parasitoids. Altogether, 145 samples were taken throughout the year: 62 samples in 37 sites in 1996, 47 samples in 30 sites in 1997, and 36 samples in 15 sites in 1998. Hosts plants were identiÞed by referring to Bonnier and de Layens (1986). Herbaceous plants, with 63 samples, included Amaranthus retroflexus L., Calendula arvensis L., Senecio vulgaris L., Sonchus sp., Taraxacum sp., Diplotaxis erucoides L. (DC.), Chenopodium glaucum L., Antirrhinum orontium L., Phaseolus vulgaris L., Malva silvestris L., Potentilla reptans L., and Solanum lycopersicum L. Woody plants, with 82 samples, were Populus nigra L., Prunus domestica L., Pyrus communis L., Pyrus malus L., Quercus coccifera L., and Q. rotundifolia Lam. Each sample consisted of 25Ð50 plants or parts of plants (branches and leaves) of the same plant species containing fresh mines or showing symptoms of leafminer presence. Olive trees (Olea europaea L.) with fruits attacked by Bactrocera oleae (Gmelin) (Diptera) were also sampled. Samples were transported to the laboratory, and leaves or parts of plants containing mines were isolated in glass vials and placed in climate-controlled chambers until adult emergence. The debris and desiccated plant material were sieved to collect the emerged leafminers and parasitoids for identiÞcation. Identification of Parasitoids. Phyllocnistis citrella parasitoids were identiÞed using the key published by Schauff et al. (1998). Other leafminer parasitoids were identiÞed using the keys of Peck et al. (1964), Askew (1968), Boucek (1988), and Goulet and Huber (1993). IdentiÞcations were made by the authors and conÞrmed by J. LaSalle (British Museum Natural History, London, United Kingdom) and by M. J. Verdu´ (Institut Valencia` d⬘Investigacions Agra`ries, Vale` ncia, Spain). Voucher specimens were deposited in the Entomology Insect Collection of the Universitat Polite` cnica de Vale` ncia, Vale` ncia, Spain. Additionally, voucher specimens of C. brevis, Pnigalio sp., and Ratzeburgiola cristata (Ratzeburg) were deposited in the

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Insect Collection of the Natural History Museum, London, United Kingdom. Data Analysis. To evaluate the inßuence of pesticides on the percentage of parasitism, the level of parasitism on P. citrella during the years 1996 and 1997 was compared among samples with and without pesticide applications in the year of sampling. No significant differences were found (percent parasitism in third and fourth instars ⫾ SE: treated orchards: 35.6 ⫾ 0.1%, n ⫽ 247; nontreated orchards: 35.2 ⫾ 0.2%, n ⫽ 169; t414 ⫽ ⫺0.172, P ⫽ 0.863). For this reason, data from treated and nontreated orchards were analyzed together. All statistical tests were performed using SAS/STAT statistical software (SAS Institute 1990). Proportions were transformed using the arcsine square-root function to stabilize variance. Changes in percentage parasitism in treated and nontreated orchards were compared using StudentÕs t-test assuming equal variance. Data on percentage of parasitism comparing each year between the four development stages parasitized, and among years in overall mean annual percentage parasitism, were analyzed by one-way analysis of variance (ANOVA) using the PROC GLM procedure and considering samples as experimental units. Means were separated using a protected least signiÞcant difference (LSD) test. The predominance of native parasitoids in different life stages was compared among years by ␹2 tests. Least squares analysis was used to Þt linear and polynomial models to evaluate the relationship between population density of citrus leafminer and level of parasitism (PROC REG procedure). F-test and r2 values for each model were evaluated. The relationship between percentage of parasitism and frequency of superparasitism and hyperparasitism was analyzed with a correlation model. Results Parasitoid Assemblage Associated with P. citrella. All of the 11,587 adult native parasitoids reared out between 1995 and 2001 belonged to two families, Eulophidae (nine species) and Pteromalidae (one species) (Table 2). The two most abundant species, Pnigalio sp. and Cirrospilus brevis Zhu, LaSalle and Huang, represented ⬎90% of the parasitoids. Secondary species, representing 7% of all the parasitoids, were Sympiesis gregori Boucek, Cirrospilus pictus (Nees), C. vittatus Walker, and Ratzeburgiola cristata. Finally, minor species (representing together ⬍1% of total parasitoids) included Neochrysocharis formosa (Westwood), Chrysocharis pentheus (Walker), Baryscapus sp., and Pteromalus sp. The relative abundance of the main parasitoids changed between 1995 and 2001 (Table 2). In 1995, Pnigalio sp. was the prevalent species and C. brevis represented only 3% of the parasitoids. C. brevis increased considerably in 1996 and reached a value of 22% of total parasitoids. From 1997 onwards, Pnigalio sp. and C. brevis coexisted in the citrus orchards of the area and were the predominant parasitoids in most

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Table 2. Relative abundance of native parasitoids of P. citrella and total no. of native parasitoids and of the introduced parasitoid C. phyllocnistoides Species Pnigalio sp. Cirrospilus brevis Sympiesis gregori Cirrospilus vittatus Cirrospilus pictus Ratzeburgiola cristata Neochrysocharis formosa Baryscapus sp. Chrysocharis pentheus Pteromalus sp. Total no. of native parasitoids Citrostichus phyllocnistoides

Species composition (%) 1995

1996

1997

1998

1999

2000

2001

Mean

89.6 3.0 0.7 3.8 3.0 0.0 0.0 0.0 0.0 0.0 605 Ñ

68.3 21.9 4.0 1.5 2.4 1.2 0.1 0.2 0.0 0.2 2,285 Ñ

30.1 65.9 1.7 0.8 0.2 0.7 0.2 0.3 0.1 0.05 2,706 Ñ

34.8 56.0 5.5 1.8 0.7 0.5 0.2 0.4 0.2 0.0 3,167 Ñ

51.3 38.3 7.0 1.5 0.7 1.0 0.1 0.0 0.0 0.0 1,630 702

23.1 67.5 4.3 2.0 1.6 1.4 0.0 0.0 0.1 0.0 1,074 8,293

53.3 30.0 3.3 10.8 0.0 2.5 0.0 0.0 0.0 0.0 120 12,237

44.8 46.9 4.1 1.7 1.1 0.9 0.1 0.2 0.1 0.1 11,587 21,232

Results of samplings made in citrus orchards in the eastern Spain region from 1995 to 2001.

samples. Secondary and minor species remained stable along the initial 5 yr of the study, except for Sympiesis gregori, which increased from 1% in 1995 to 7% in 1999. In 2000 and 2001, when the introduced parasitoid C. phyllocnistoides dispersed and established all over the area, the number of native parasitoids found on P. citrella decreased dramatically, representing 11.4 (2000) and 1% (2001) of the total parasitoid assemblage (Table 2). Only the two most abundant and some secondary species remained attacking P. citrella. Abundance of Native Parasitoids in Other Leafminers. The leafminers found attacking herbaceous plants were all unidentiÞed Agromyzidae (Diptera), whereas those attacking trees were Lepidoptera [mostly Stigmella malella (Stainton), Leucoptera malifoliella (O. G. Costa), and Phyllonorycter blancardella (Fab.)]. On herbaceous plants, the most common leafminer parasitoids were Diglyphus minoes (Walker) (44.1%), C. pubicornis (Zetterstedt) (25.8%), and C. pentheus (16.4%). On woody plants the genus Cirrospilus was widespread and included three species: C. brevis (27.0%), C. variegatus (Massi) (24.3%), and C. vittatus (13.0%) (Table 3). The relative abundance of these species was quite similar over the 3-yr study period except for C. brevis, which increased its presence on tree leafminers from 4.5% in 1996 to 29.6% in 1998.

These results showed that the proportion of leafminer parasitoids capable of attacking P. citrella was higher in woody (69.7%) than in herbaceous plants (22.2%). The two major native parasitoids found on P. citrella (Pnigalio sp. and C. brevis) represented 31.3% of the parasitoids on trees (appearing on leafminers from all the woody plants sampled except Quercus) and only 2.2% of total parasitoids found on herbs (appearing only in leafminers of Sonchus sp. and Potentilla reptans). Host Stage Preference of Native Parasitoids. Parasitoid incidence in all susceptible stages of P. citrella was studied from a total of 5,508 parasitoids reared individually to adult stage. There were differences in host stage preference among species of parasitoids (Fig. 1). All species developed mostly from third and fourth instars of P. citrella, with three species predominating among those reared from fourth instars (Pnigalio sp., Sympiesis gregori, and Ratzeburgiola cristata) and three Cirrospilus species (C. brevis, C. vittatus, and C. pictus) chießy from third instars. Among minor species, Þve Neochrysocharis formosa and four C. pentheus developed from third instars and six Baryscapus sp. developed from fourth instars. The predominance of parasitoid species in different life stages of the citrus leafminer remained stable over the years (Pnigalio sp., ␹29 ⫽ 15.33, P ⬎ 0.05; C. brevis, ␹29 ⫽

Table 3. Relative abundance of hymenopterous parasitoids of leafminers collected in herbaceous and woody plants in the vicinity of citrus orchards Parasitoid species Diglyphus minoes Chrysocharis pentheus Chrysocharis pubicornis Cirrospilus brevis Cirrospilus variegatus Cirrospilus vittatus Diglyphus isaea Neochrysocharis formosa Pnigalio sp. Other hymenoptera Total

Number of parasitoids Herbs

Trees

Total

161 60 94 4 Ñ 11 22 2 4 7 365

Ñ 37 Ñ 50 45 24 Ñ 10 8 11 185

161 97 94 54 45 35 22 12 12 18 550

Result of 145 samplings made in the eastern Spain citrus region between 1996 and 1998.

Host Diptera Lepidoptera/Diptera Diptera Lepidoptera/Diptera Lepidoptera Lepidoptera/Diptera Diptera Lepidoptera/Diptera Lepidoptera/Diptera Lepidoptera/Diptera

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Fig. 1. Percentage of P. citrella life stages attacked by different native parasitoid species. Data from samplings made in the eastern Spain citrus region between 1995 and 1999.

14.16, P ⬎ 0.05; C. vittatus, ␹29 ⫽ 2.88, P ⬎ 0.05; C. pictus, ␹29 ⫽ 2.93, P ⬎ 0.05; R. cristata, ␹29 ⫽ 2.54, P ⬎ 0.05; S. gregori, ␹29 ⫽ 7.83, P ⬎ 0.05). Given the relative abundance of the parasitoid species in the Þeld and their predominance, 98% of the parasitoids were C. brevis when second instars were found to be parasitized; for third instars, 72% were C. brevis and 24% were Pnigalio sp.; for fourth instars, 64% were Pnigalio sp. and 30% C. brevis; and for pupae, 93% were Pnigalio sp. Level of Native Parasitism. A total of 93,846 live immature stages of the citrus leafminer were observed between 1995 and 1999, of which 21,460 (22.9%) were found to be parasitized. The annual mean parasitism rate, considering all susceptible development stages of P. citrella, was similar from 1995 to 1999, with no signiÞcant differences between years (F ⫽ 0.56; df ⫽ 4,715; P ⫽ 0.689), and with a mean value for the 5 yr of 22.6 ⫾ 0.6 (n ⫽ 720). During these years, the leafminer population was high and stable (Garcõ´a-Marõ´ et al. 2003). Parasitism changed with the stage of P. citrella (Fig. 2). In general, the preferred stages for parasitism were third (27Ð38%) and fourth instars (30 Ð53%). Second instars were less frequently parasitized (8 Ð 14%), and parasitism of this instar was not detected until 1997, when C. brevis increased its presence. Pupal parasitism was low (4 Ð 6%). In 1995 and 1996, the percentage of parasitism was signiÞcantly higher in fourth instars than in third instars, but from 1997 to 1999, this difference disappeared because of the relative increase of C. brevis, which parasitized mostly third instars, and a parallel decrease of Pnigalio sp., which parasitized mostly fourth instars.

The relationship between population density of citrus leafminer (measured as mines per leaf) and level of parasitism caused by native parasitoids depended on the development stages of P. citrella (Fig. 3). Parasitism inßicted by native parasitoids on third and fourth instars rose as citrus leafminer density increased from 0 to 2.5 mines per leaf and decreased at higher leafminer densities (third instar: r2 ⫽ 0.9527, n ⫽ 6, P ⬍ 0.05; fourth instar: r2 ⫽ 0.9304, n ⫽ 6, P ⬍ 0.05). For second instars, the percentage of parasitism decreased lineally as host population increased (r2 ⫽ 0.7876; F ⫽ 14.83; df ⫽ 1,4; P ⫽ 0.018). Finally, host density had little inßuence on pupal parasitism (r2 ⫽ 0.6194; F ⫽ 6.51; df ⫽ 1,4; P ⫽ 0.061). Superparasitism and Hyperparasitism. Cases of superparasitism and hyperparasitism were generally uncommon. Of 21,460 parasitized stages, the number of cases of superparasitism was 264 (1.2%), and the number of cases of hyperparasitism was 68 (0.32%). This suggests that parasitoids are able to discriminate between parasitized and unparasitized hosts and select unparasitized ones for egg laying. Most of the super- or hyperparasitoids (80%) belonged to the genus Cirrospilus. We found as superparasitoids 24 C. brevis, 5 C. vittatus, 8 Pnigalio sp., and 2 S. gregori. We found as hyperparasitoids 15 C. brevis, 2 C. vittatus, and 6 Pnigalio sp. Host stage had little inßuence on superparasitism (1.4% in second instars, 1.5% in third instars, 0.7% in fourth instars, and 1.2% in pupae), whereas hyperparasitism seemed to increase somewhat with host stage (0% in second instars, 0.38% in third instars, 0.39% in fourth instars, and 0.76% in pupae).

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Fig. 2. Mean ⫾ SE annual percentage of parasitism for each susceptible stage of P. citrella. Result of 974 samplings made in the eastern Spain citrus region from September 1995 to December 1999 (one-way ANOVAÑ1995: F ⫽ 24.44; df ⫽ 2,94; P ⬍ 0.0001; 1996: F ⫽ 154.55; df ⫽ 2,630; P ⬍ 0.0001; 1997: F ⫽ 158.96; df ⫽ 3,757; P ⬍ 0.0001; 1998: F ⫽ 78.44; df ⫽ 3,508; P ⬍ 0.0001; 1999: F ⫽ 30.39; df ⫽ 3,297; P ⬍ 0.0001). For each year, different letters are signiÞcantly different according to an LSD test (P ⱕ 0.05). The arcsine of the square root of the percent parasitism was taken to normalize the variation around the mean.

Superparasitism and hyperparasitism were most frequent in samples with a high percentage of parasitism. When parasitism was ⬎90%, the percentage of samples with at least one case of superparasitism was close to 70% and the percentage of samples with at least one case of hyperparasitism was ⬇40% (Fig. 4). The occasional appearance of super- and hyperparasitism thus seems to be related to extreme cases of intra- and interspeciÞc competition that occur rarely in the Þeld. Discussion This study shows how native species of leafminer parasitoids moved on the new host shortly after the

introduction of citrus leafminer in the Valencia area in 1994, and within a few years a parasitoid assemblage of 10 species built up around the invading herbivore. The parasitoid species complex preying on the citrus leafminer was similar all around the Mediterranean basin and different from the parasitoid complex found in other areas of invasion such as Australia, the Americas, and South Africa (Schauff et al. 1998), reßecting differences in the pool of species available. The same species of citrus leafminer parasitoids reported in this study of the Valencia area were found in samples collected in other areas of continental Spain and the Balear Islands between 1996 and 1999 (unpublished data). A very similar species complex

Fig. 3. Regression analysis of the percentage of parasitism (y) as a function of host density (x, number of mines per leaf) in P. citrella. Data from samplings made in the eastern Spain citrus region between 1996 and 1999 (modelsÑsecond instars: r2 ⫽ 0.7876, y ⫽ 16.919 ⫺ 2.3782x; third instars: r2 ⫽ 0.9527, y ⫽ ⫺1.234x2 ⫹ 4.537x ⫹ 34.064; fourth instars: r2 ⫽ 0.9304, y ⫽ ⫺2.5429x2 ⫹ 13.703x ⫹ 25.919; pupae: r2 ⫽ 0.6194, y ⫽ 0.558x ⫹ 3.8736).

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Fig. 4. Percentage of parasitism in samples (x) versus percentage of samples with at least one case of superparasitism or hyperparasitism (y). Result of 974 samplings made in the eastern Spain citrus region from September 1995 to December 1999 (superparasitism: r2 ⫽ 0.8960, n ⫽ 5, y ⫽ 5.4248*e0.0299x; hyperparasitism: r2 ⫽ 0.9671, n ⫽ 5, y ⫽ 0.0093x2 Ð0.4346x ⫹ 5.3846).

was also reported by other authors in surveys carried out in smaller areas and/or over shorter sampling periods in other Spanish regions (Gonza´lez et al. 1996, Lacasa et al. 1997, Ma´rquez et al. 2003). In the Canary Islands, we found two more species: Chrysocharis gemma (Walker) and Achrysocharoides sp. (unpublished data). These Þndings agree that two species stand out as the most abundant and widespread attacking the citrus leafminer in Spain: Pnigalio sp. and C. brevis. C. brevis, formerly treated in the literature as Cirropilus sp. near lyncus or Cirrospilus sp. D (Schauff et al. 1998), was recently described by Zhu et al. (2002) from the analysis of material collected in our study. Earlier reports of this species are from China (Sheng and Wang 1992, as C. lyncus), Japan (Heppner 1993, Ujiye and Adachi 1995, as Cirrospilus sp.), and Taiwan (Ujiye et al. 1996, as C. sp. near lyncus), always with citrus leafminer as host. The process of parasitoid recruitment in Spain was very rapid. We observed little parasitism in the very Þrst months immediately after the rapid colonization of the citrus leafminer in the area (autumn of 1994 to summer of 1995; unpublished data). However, in autumn of 1995, 1 yr after the arrival of the citrus leafminer in eastern Spain, Þve species of parasitoids were already found on P. citrella. Pnigalio sp. was the Þrst species to adapt to the new host, establishing quickly as the main parasitoid in all the orchards in 1995. Studies made the same year in another part of Spain (Huelva) showed similar results (Gonza´lez et al. 1996). In contrast, C. brevis required 3 yr to become one of the two major parasitoids of the citrus leafminer. Two hypotheses could be given to explain this delay in the recruitment of C. brevis. First, C. brevis was present as a native parasitoid at very low population levels when the citrus leafminer arrived and

needed some time to build up in numbers and/or to adapt to the new host. Second, C. brevis was not a native species but arrived together with the citrus leafminer, needing some time to complete its expansion and to adapt to the environmental conditions of the new area. In this study, C. brevis was found parasitizing other leafminers in the area, but it was never identiÞed in earlier surveys in Spain, neither on Agromyzid leafminers in the Valencia area (Verdu´ 1991, France´ s 1994), nor on apple leafminers in Lleida (Olivella 1996, Bellostas et al. 1998), raising doubts about its presence in Spain before the introduction of the citrus leafminer. Between 1996 and 1998, we observed the relative increase of C. brevis as a parasitoid of citrus leafminer and other tree leafminers in the area, suggesting a recent introduction. Other studies have also reported cases of parasitoids invading new areas after their hosts (Godfray et al. 1995). A further genetic study of the Asian and Mediterranean populations would help distinguish if C. brevis is an invader or native species in the Mediterranean area. Eight of the 10 species found in our study represented ⬍10% of the total native parasitoid species reared from citrus leafminer. Cornell and Hawkins (1993) indicated that when invading hosts reach high densities in the region of introduction, minor species of parasitoids may represent incidental attacks by nonadapted native parasitoids simply because the new host is abundant. Such incidental attacks would not occur at lower, more normal population densities of the new host. This is what apparently occurred in Spain when the exotic parasitoid C. phyllocnistoides was introduced, becoming widespread and lowering citrus leafminer populations from the year 2000 onward (Garcõ´a-Marõ´ et al. 2003). In these circumstances, only the two major species of native parasi-

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toids (Pnigalio sp. and C. brevis) and a few secondary species remained attacking the citrus leafminer. The native parasitoid species that act on invading herbivores are likely to be those that attack similar hosts with similar ecology (analogous taxonomic group, feeding niche, food plants) (Askew and Shaw 1986, Godfray 1994). The results of our analysis seem to follow this trend. Two of the four main parasitoids of Lepidopteran leaf-mining insects on trees (C. brevis and C. vittatus) were common on the citrus leafminer, whereas the most abundant parasitoids attacking Dipteran leafminers of herbaceous plants (Diglyphus minoes, D. isaea, and C. pubicornis) never attacked the citrus leafminer or did it only in very low numbers (C. pentheus). The fact that Pnigalio sp., considered as a secondary parasitoid on native tree leafminers, was the Þrst to attack the invading host and became its major parasitoid is also remarkable. It shows how minor and secondary natural enemies of native hosts can play a major role on introduced herbivores. These results document the beneÞts of preserving biodiversity (LaSalle 1993), stressing how native fauna has potential value as initial suppliers of biological control agents for introduced herbivores and can also serve as a seasonal reservoir of natural enemies (Massa et al. 2001, Rizzo 2003). The predictions of Cornell and Hawkins (1993) for parasitoid accumulation on herbivores in invasion areas compared with native areasÑless species richness, more generalist species, and lower percentage of parasitismÑ can be assessed in the process of expansion exhibited by the citrus leafminer from the early 1990s. The number of citrus leafminer parasitoids found in the three countries of Asia with the highest number of reported species was 23 in Japan, 17 in Thailand, and 15 in China (Schauff et al. 1998). We identiÞed 10 species in our survey. Two other Mediterranean countries with high number of species were Cyprus (11) and Italy (10). The three countries of the Americas with the highest number of reported species attacking this introduced pest were the United States (13), Mexico (12), and Honduras (10) (Schauff et al. 1998). Another feature reportedly observed in native parasitoid complexes of invading herbivores is the generalized habits of the parasitoids (Cornell and Hawkins 1993). All native parasitoids of the citrus leafminer found in our study are polyphagous species with idiobiont strategy. All of them are also ectoparasitoids, with the exception of the endoparasitoids C. pentheus and N. formosa. Similar results were found in other areas where this pest has invaded (Schauff et al. 1998). Our study also shows that the level of parasitism inßicted by native parasitoids was low to moderate, as reported in other Mediterranean countries (Malausa et al. 1996, Uygun et al. 1996, Argov et al. 1998, Siscaro et al. 2003). In the native area of the pest, parasitism reaches between 30 and 70% (Ujiye and Adachi 1995, Morakote and Nanta 1996, Tan and Huang 1996). The low percentage of parasitism often observed in parasitoid complexes on invading hosts is usually attributed to the lack of adaptation of the native species to the new host, but it could also be caused, at least in

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part, by density effects as well, because many parasitoids exhibit negative density dependence at very high host density (Cornell and Hawkins 1993). Our results show that, in the two main stages attacked by parasitoids (third and fourth instars), parasitism rates increased with host population abundance at low to moderate population levels, but decreased when host population reached higher levels, ⬎2.5 mines per leaf. These Þndings suggest that citrus leafminer escapes the action of the parasitoids at high population densities. Between 1995 and 1999, population levels attained by the citrus leafminer were very elevated during summer and autumn in eastern Spain, despite the numerous parasitoid species that attacked the new host. This would suggest that the native parasitoid assemblage was not effective enough to maintain the invading host at low population levels. Only after establishment of the exotic parasitoid C. phyllocnistoides did a sharp decrease in host density occur (Garcõ´aMarõ´ et al. 2003). In summary, after the invasion of citrus leafminer in Spain, native parasitoids species moved onto the new host, with the assemblage following typical patterns for native parasitoid complexes attacking invading pestsÑlower parasitoid species richness, generalized host ranges, idiobionts, and low to moderate rates of parasitism in the Þeld. The high population level reached by the new pest, associated with a negative density dependent response of the parasitoids at these high population levels, suggests that the parasitoid assemblage played only a limited role in regulating the population of the new host and justiÞes the program of classical biological control developed to introduce new species of parasitoids. Acknowledgments We thank C. Alfaro, A. Alonso, V. Bueno, D. Castrillo´ n, M. Villalba, C. Granda, C. Moreno, J. M. Rodriguez, and E. Sanz for technical assistance in many aspects of the project, including sampling and insect rearing. We are indebted to P. A. Stansly (University of Florida) for comments and constructive suggestions on the manuscript. Financial support was provided by Consellerõ´a d⬘Agricultura (project GV-CAPA00-18) and Consellerõ´a de Cultura, Educacio´ i Cie` ncia (project GV-D-AG-01-128-96) of the Generalitat Valenciana, and Ministerio de Agricultura, Pesca y Alimentacio´ n (project INIA SC95-108-C5-4) and Ministerio de Educacio´ n y Cultura (project CICYT AGF97-0899-C02-02).

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leafminer in Turkey. Proceedings of the International Conference of managing the citrus leafminer, 22Ð25 April 1996, Orlando, FL. Vercher, R. 2000. Control biolo´ gico del minador de hojas de cõ´tricos Phyllocnistis citrella (Lep: Gracillariidae). PhD dissertation, Universidad Polite´ cnica de Valencia, Valencia, Spain. Vercher, R., F. Garcia-Marı´, J. Costa-Comelles, C. Marzal, and C. Granda. 2000. Importacio´ n y establecimiento de para´sitos del minador de hojas de cõ´tricos Phyllocnistis citrella (Lepidoptera: Gracillariidae). Bol. San. Veg. Plagas. 26: 577Ð591. Verdu´ , M. J. 1991. Chalcidoidea (Hym, Apocrita, Terebrantia) en plantas hortõ´colas de la Comunidad Valenciana. Bol. Asoc. Esp. Entom. 15: 245Ð255. Zhu, C. D., J. Lasalle, and D. W. Huang. 2002. A study of chinese Cirrospilus Westwood (Hymenoptera: Eulophidae). Zool. Studies. 41: 23Ð 46. Received for publication 27 December 2004; accepted 2 August 2005.