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Biological Control 52 (2010) 37–45
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Biological Control
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Changes in predation and parasitism of the citrus leafminer Phyllocnistis citrellaStainton (Lepidoptera: Gracillariidae) populations in Spain followingestablishment of Citrostichus phyllocnistoides (Hymenoptera: Eulophidae)
F. Karamaouna a, S. Pascual-Ruiz b, E. Aguilar-Fenollosa b, M.J. Verdú c, A. Urbaneja c, J.A. Jacas b,*
a Benaki Phytopathological Institute, 8 Stefanou Delta Street, 14561-Kifissia, Athens, Greeceb Unitat Associada d’Entomologia Agrícola Universitat Jaume I (UJI), Departament de Ciències Agràries i del Medi Natural, Campus del Riu Sec, E-12071 Castelló de la Plana, Spainc Instituto Valenciano de Investigaciones Agrarias (IVIA), Ctra. Montcada-Nàquera km 4.5, E-46113 Montcada, Spain
a r t i c l e i n f o a b s t r a c t
Article history:Received 13 January 2009Accepted 15 September 2009Available online 19 September 2009
Keywords:Phyllocnistis citrellaCitrostichus phyllocnistoidesPost-release evaluationClassical biological controlCitrus
1049-9644/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.biocontrol.2009.09.009
* Corresponding author. Fax: +34 964728216/34 96E-mail address: [email protected] (J.A. Jacas).
The citrus leafminer, Phyllocnistis citrella, is a pest native to Southeast Asia which threatened the citrusindustry in the Mediterranean region upon its introduction in 1993. Immediately afterward, a classicalbiological control program was implemented in Spain. The exotic parasitoid Citrostichus phyllocnistoideswas the only introduced parasitoid to become established. In 2006, data on both the incidence of P. citrellaand the impact of its natural enemies were collected following the same protocols used in 1997–1999when C. phyllocnistoides was not yet present. C. phyllocnistoides constituted 99.4% of the parasitoids col-lected in 2006 corresponding to a decrease in the incidence of P. citrella from 3.2–5.1 to 1.8–2.4 mines perleaf in 1997–1999 and 2006, respectively. Mortality caused by natural enemies on P. citrella in 2006 was93.3% (18.0% parasitism, 40.8% feeding punctures and 34.5% predation). C. phyllocnistoides, which prefer-entially parasitizes P. citrella second instar larvae, has displaced most of the indigenous parasitoids thatmoved onto P. citrella mainly parasitizing third instar larvae, upon its introduction. Because C. phyllocn-istoides is an idiobiont parasitoid and preferentially parasitizes P. citrella second instars, this stage hasbecome dominant in the orchards. The shift in the relative abundance of P. citrella larvae has promptedgeneralist predators to prey mostly on second instars and has contributed to the displacement of thenative non-specific parasitoids, which principally utilize third instars, from the system. Both indigenouspredators and the introduced parasitoid are key players in the natural regulation of P. citrella.
� 2009 Elsevier Inc. All rights reserved.
1. Introduction
The citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepi-doptera: Gracillariidae) is a citrus pest native to southeastern Asiathat has spread to most citrus growing areas in the Mediterraneanand the Americas during the last decade of the 20th century(Urbaneja et al., 2000). Studies carried out between 1996 and1999 showed that in Mediterranean areas, CLM damaged only 5–15% of the annual new leaf area of mature trees and therefore yieldwas not affected by the pest (Garcia-Mari et al., 2002). However, itis still regarded as an important pest in nurseries, as well as onyoung plants and top-grafted trees, where chemical control is nec-essary (Garcia-Mari et al., 2002).
As in similar situations of exotic pest outbreaks (Michaud,2002), the rapid spread of CLM in Spain in 1995 prompted fundingagencies to prioritize biological control projects based on the rear-ing and release of imported natural enemies. CLM became a target
ll rights reserved.
3424001.
of the classical biological approach without a critical evaluation ofwhether such an approach was the best one. In Spain, pre-intro-duction studies of natural enemies of CLM (Lenteren and Woets,1988; Barbosa and Segarra-Carmona, 1993; FAO, 1996; EPPO,1999, 2000) were very limited (Urbaneja et al., 2000, 2003a), andsix different parasitoids were introduced in less than 3 years (from1996 to 1998) (Jacas et al., 2006). The host-specific endoparasitoidAgeniaspis citricola Logvinovskaya (Hymenoptera: Encyrtidae) suc-cessfully established in the Canary Islands, whereas the oligopha-gous facultative hyperparasitoid Citrostichus phyllocnistoides(Narayan) (Hymenoptera: Eulophidae) established in mainlandSpain (Garcia-Mari et al., 2004). This species was also introducedand established in other Mediterranean countries (Argov and Ross-ler, 1996, 1998; Tsagarakis et al., 1999; Rizqi et al., 2003; Siscaroet al., 2003). Currently, CLM is no longer considered an importantpest of citrus on mature bearing trees (Garcia-Mari et al., 2002).
Three years after the introduction of CLM in Spain, a 3-year sur-vey (1997–1999) carried out in the region around the city of Valè-ncia (Fig. 1) – the main citrus growing area in Spain – showed thatindigenous natural enemies of CLM, consisting of 11 generalist
Montcada
L’Alcúdia
LlíriaValència
Les Alqueries
Elx
BéteraMontcada
L’Alcúdia
LlíriaValència
Les Alqueries
Elx
Montcada
L’Alcúdia
LlíriaValència
Les Alqueries
Elx
Bétera
Fig. 1. Location of the clementine orchards considered in this study (Bétera,L’Alcúdia, Les Alqueries, Llíria, Montcada) and in the 1997–1999 survey (Montcada,Elx).
38 F. Karamaouna et al. / Biological Control 52 (2010) 37–45
parasitoids and several unidentified polyphagous predators(including lacewings, spiders), fed on CLM populations resultingin up to 70% mortality of mature larvae by the end of the summer(Urbaneja et al., 2000). Predation was the main mortality factor(35.4%), followed by parasitoid host feeding (20.3%) and parasitism(16.9%). Composition of the parasitoid species complex changedduring the 3 years of the study. However, the indigenous eulophidsCirrospilus brevis (=ca. lyncus) Zhu, LaSalle and Huang and Pnigaliopectinicornis L. were consistently the predominant species whereasthe exotic parasitoids introduced up until then either had no signif-icant effect on CLM populations (the eulophids Quadrastichus citrel-la Girault and Galeopsomyia fausta LaSalle) or did not succeed inoverwintering (A. citricola). Studies carried out during the releaseand establishment of C. phyllocnistoides from 1998 to 2001 (Gar-cia-Mari et al., 2004) showed a shift in parasitoid species composi-tion: C. phyllocnistoides represented 97.1% of parasitoids in 2001.This change affected percentage parasitism, which according tothat study increased from 20–25% in 1995–1998 to near 60% in1999–2001. Such changes apparently affected mean CLM densities(34% and 72% reductions for eggs and adults, respectively) andfoliar damage (56% reduction). However, no comparison of totalnatural enemy-caused mortality before and after the establish-ment of C. phyllocnistoides has been undertaken. Successful controlof a pest by natural enemies (either exotic or indigenous) must bedetermined by quantitative evaluation both before and after re-lease (DeBach et al., 1976; Luck et al., 1988). For this reason thepresent study was carried out. Our aims were to monitor the inci-dence of CLM 12 years after its arrival, to quantify the impact of itsnatural enemies (both indigenous and exotic parasitoids and pre-dators) in citrus orchards of the València region and to comparethese results to those obtained before the establishment of C. phyl-locnistoides. This should allow us to evaluate the biological controlprogram against CLM in Spain.
2. Materials and methods
The methodology used was based on the survey carried outfrom 1997 to 1999 (Urbaneja et al., 2000). Hence, leaf flushing pat-terns and incidence of both the CLM and its parasitoids wererecorded at 7–15 day intervals from early June until December2006 in three citrus orchards at three different locations in theregion of València: L’Alcúdia, Bétera and Llíria (Fig. 1). These orch-ards were 1 ha each and consisted of mature bearing clementinetrees [Citrus reticulata Blanco] grafted on Carrizo Citrange rootstock
[Poncirus trifoliata (L.) Rafinesque-Schmaltz � Citrus sinensis (L.)Osbeck]. The orchards at L’Alcúdia and Bétera received no chemicaltreatments during the sampling period except for a mineral oilapplication at L’Alcúdia against Aonidiella aurantii (Maskell)(Hemiptera: Diaspididae) on August 24, 2006. The orchard at Llíriafollowed IPM guidelines and was treated with pirimicarb againstAphis spiraecola Pagensteche (Hemiptera: Aphididae) on August 3and with methylpyrimiphos against A. aurantii on August 10. Allthree orchards were drip irrigated with a ground cover of Festucaarundinacea Schreber (Poaceae) that was periodically mowed andwere surrounded by similar citrus orchards.
Twenty trees were randomly chosen at each sampling date. Aring made of flexible polyethylene irrigation tubing, 56 cm indiameter, was thrown onto the tree and the number of total leafflushes, egg-receptive flushes (those exhibiting leaves up to 2 cmlong) and flushes containing CLM larvae or pupae found withinthe ring were counted. Three additional flushes, or suckers whenno new shoots were available, were collected from each tree, putinto a plastic bag and transported to the laboratory in a coolerfor observation of their infested leaves under a stereoscopic binoc-ular microscope. Leaves were examined for the presence of mines(either occupied or abandoned), CLM larvae (first to fourth instarsbased on their morphology) or pupae and parasitoid immaturestages. Observations were terminated once 100 mines had been in-spected. Dead CLM stages exhibiting black spots were consideredthe product of either host feeding by parasitoids or predation byinsects with sucking mouthparts. Predation was assumed to bethe reason for incomplete mines or mines where mutilated CLMlarvae were found. Because both indigenous and naturalized para-sitoids of CLM are idiobiont species that cause permanent paralysiswhen stinging the host, the instar of parasitized hosts wasrecorded to establish parasitoid ovipositional preferences. Basedon these data, percentage parasitism, host feeding and predationwere calculated (number of individuals either parasitized, piercedor preyed upon, respectively, divided by the total number of indi-viduals inspected). Data for stage-specific natural enemy-causedmortality were combined with the actual distribution of the differ-ent stages observed in the field and used to estimate average sur-vival of CLM immature stages. When possible, results werecompared to those collected from 1997 to 1999 in Elx and Montca-da (Fig. 1) by Urbaneja et al. (2000) although these authors did notconsider CLM first instars in their study.
About 100 supplementary fully grown infested flushes werecollected at the three orchards described above at each visit andat two additional clementine orchards located at the InstitutoValenciano de Investigaciones Agrarias (IVIA, Montcada; 10 sam-ples between 4 August and 4 December 2006) and Les Alqueries(3 samples during August 2006) (Fig. 1) at different visits. Theseflushes were transported to the laboratory in a cooler where theywere further enclosed in cardboard cages (22 � 30 � 22 cm). Aclear PVC vial (1 cm diam 10 cm long) was inserted in one of thesides of the cages and used as an emergence trap for parasitoids,which were prepared for taxonomic determination afteremergence.
3. Results
The three orchards were sampled 16 times between June 15(date when infested leaves were first observed in Bétera) andDecember 28 (when infested leaves were last observed in bothBétera and L’Alcúdia). A total of 3548 mines were found in 2142infested leaves inspected. Of the mines found, 1609 mines werecomplete (including empty mines left behind by emerged CLM)and therefore were considered to house apparently healthy CLMlarvae and pupae. Additionally, 545 parasitized individuals
F. Karamaouna et al. / Biological Control 52 (2010) 37–45 39
(15.4%), 724 punctured larvae (20.4%) and 670 immature stagesexhibiting evidence of predation (18.9%) were observed in theseleaves. Caged leaves from the samples taken at the five locations(Fig. 1) yielded a total of 3590 adult wasps. These specimens wereidentified (Table 1) and C. phyllocnistoides was the predominantspecies representing 99.4% of the total.
3.1. Incidence of the citrus leafminer at orchard sites
The CLM first appeared in the orchard at Bétera on June 15(Fig. 2a), then at L’Alcúdia on the 29 (Fig. 2b) and finally at Llíriaon July 13 (Fig. 2c). At all locations the percentage of infestedflushes rapidly increased and reached values close to 100% in about4 weeks from detection. These high values remained fairly con-stant at both Llíria and Bétera, where an average of 15–20 newflushes per m2 appeared during the summer period of July–Sep-tember. However, the percentage of flushes per m2 suddenlydropped to near zero at L’Alcúdia from August 10 until September21. New flushes became infested as soon as they appeared but theoverall percentage of infested flushes at L’Alcúdia remained low(<20%) for the rest of the season in coincidence with the oil treat-ment applied in that orchard at the end of August. At Llíria, thecoldest location surveyed, infestation started declining on Septem-ber 7 (1 month after the pesticide treatments against aphids andscales) and on October 3 at Bétera. By the second half of Decemberthe CLM became undetectable in all three orchards.
During the study period, CLM densities (both healthy and thoseattacked by natural enemies) ranged from 0.69 to 4.35 mines perleaf at Bétera (mean: 2.40 ± 0.30) (Fig. 3a), from 0.99 to 5.00 minesper leaf at L’Alcúdia (mean: 2.14 ± 0.28) (Fig. 3b), and from 0.55 to3.61 mines per leaf at Llíria (mean: 1.79 ± 0.24) (Fig. 3c). Maximumvalues occurred between August 22 and September 18 at bothL’Alcúdia and Llíria (5.0 and 3.6 mines per leaf, respectively),whereas at Bétera, three similar peaks of around 4 mines per leafwere detected between July 27 and September 7.
3.2. Incidence of CLM natural enemies
The number of parasitoids per infested leaf ranged from 0.03 to0.71 in Bétera (mean: 0.40 ± 0.07), from 0.01 to 0.91 in L’Alcúdia(mean: 0.33 ± 0.07), and from 0.01 to 1.18 in Llíria (mean:0.25 ± 0.11) (Fig. 3). Trends in parasitism followed CLM popula-tions with maximum values reached at the beginning of Septemberin both L’Alcúdia (September 5) and Llíria (September 7), but morethan 1 month later in Bétera (October 19) (Fig. 3a–c). The com-bined impact of parasitoids and predators increased as the seasonprogressed and reached maximum values of 83–96% mortality atthe end of August in all three locations (Fig. 4). Thereafter, mortal-ity due to biological control slowly decreased until October, whenit increased again at both L’Alcúdia and Bétera, the organic orch-ards, and fluctuated around 50% until the end of the season
Table 1Composition of citrus leafminer parasitoid guilds (actual counts and percentages) in the d
Parasitoid species L’Alcúdia Bétera Llíria
n % n % n
Citrostichus phyllocnistoides 828 100.0 1250 99.6 591Cirrospilus brevis 0 0.0 0 0.0 0Pnigalio spp. 0 0.0 3 0.2 4Sympiesis gregori 0 0.0 2 0.2 0Pteromalidae sp. 0 0.0 0 0.0 4Diglyphus sp. 0 0.0 0 0.0 1
Total 828 1255 600
(Fig. 4a and b). In Llíria, natural enemy-caused mortality showeda steady decline until December (Fig. 4c).
3.3. Parasitic complex composition and CLM stage incidence
Composition of the parasitoid complex developing on the CLMwas almost identical at all locations sampled (Table 1). C. phyllocn-istoides always represented more than 96.7% of total specimensexamined. Also five indigenous parasitoids (the eulophids C. brevis,Pnigalio sp., Sympiesis gregori Boucek, Diglyphus sp. and an undeter-mined pteromalid species) were recorded and each of them repre-sented less than 2.2% of the total. Oviposition on second instarlarvae of CLM was the highest (67.5%), followed by third instars(31.5%) (Fig. 5). For second instars to pupae, data on the relativecontribution of natural enemies to stage specific mortality isshown in Fig. 6. Mean mortality from first instar to pupa in 2006resulted 93.3%.
In 1997–1999, the highest percentages of predation, ovipositionand feeding punctures were recorded on CLM third instars (71.3%,66.5%, and 74.4%, respectively) (Fig. 5) whereas in 2006 the maxi-mum values of predation, oviposition and feeding punctures wererecorded on CLM second instars (52.9%, 67.5%, 80.4%, respectively)(Fig. 5). According to our results, mortality inflicted by natural ene-mies on the CLM from the second instar to adulthood has increasedfrom 72.6% (16.9% parasitism, 20.3% feeding punctures and 35.4%predation, Fig. 6a) in 1997–1999 to 89.0% (29.4% parasitism,33.2% feeding punctures and 26.5% predation, Fig. 6b) in 2006 afterthe establishment of C. phyllocnistoides (Fig. 6). This additional16.4% in mortality was mainly due to the enormous increase ofparasitism (from 0.4% to 21.9%), feeding punctures (from 7.3% to28.3%) and predation (from 6.9% to 16.5%) on CLM second instar(Fig. 6). On average, less than half of the CLM that completed thesecond instar and entered the third instar before the introductionof C. phyllocnistoides, could successfully molt into this stage in2006. Furthermore, mortality of CLM third instar also increasedconsiderably (from 39.6% to 66.0%) and data on predation andfeeding punctures on CLM first instar that are missing for the1997–1999 period demonstrate that this stage suffered on averagea 40% reduction in 2006 (20.4% feeding punctures, 18.2% predation,no parasitism). Neither predators nor parasitoids substantiallyaffected CLM fourth instars and pupae, so the overall number ofindividuals successfully developing from the second instar toadulthood in 2006 was almost one third of that registered in1997–1999.
4. Discussion
The results obtained at the three locations sampled in 2006show that incidence of the CLM has decreased upon establish-ment of the exotic parasitoid C. phyllocnistoides. The percentagesof infested shoots recorded during 1997–1999 (Urbaneja et al.,2000) and in 2006 were similar at the beginning of the season,
ifferent Clemenules orchards included in this survey.
Les Alqueries Montcada (IVIA) Total
% n % n % n % Total
98.5 89 96.7 811 99.5 3569 99.40.0 1 1.1 1 0.1 2 <0.10.7 2 2.2 3 0.4 12 0.30.0 0 0.0 0 0.0 2 <0.10.7 0 0.0 0 0.0 4 0.10.2 0 0.0 0 0.0 1 <0.1
92 815 3590
BÉTERA
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a
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Fig. 2. Flushing patterns (number of flushes per m2) and incidence of citrus leafminer in three different clementine orchards (a, Bétera; b, L’Alcúdia; c, Llíria) along 2006.Receptive flush refers to flushes exhibiting leaves smaller than 2 cm, which are preferred over older leaves for oviposition.
40 F. Karamaouna et al. / Biological Control 52 (2010) 37–45
steadily increasing to approximately 100% in August. Almostevery new shoot appearing afterward was infested in the 1997–1999 study, whereas this percentage fell to almost 20% fromthe end of summer (August) until the end of the season (Decem-
ber) in 2006 (Fig. 2). The oil applied at the end of August inL’Alcúdia may have contributed to the low percentage of infestedflushes detected in that orchard after the treatment until the endof the season, as oils can prevent CLM oviposition and have an
15-J
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# Parasitized CLM # Pierced CLM # Mutilated CLM # Healthy CLM
a
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Fig. 3. Incidence of citrus leafminer (number of individuals per leaf) and its natural enemies (number of citrus leafminer individuals exhibiting signs of predation, feedingpunctures, or parasitism) in three different clementine orchards (a, Bétera; b, L’Alcúdia; c, Llíria) along 2006.
F. Karamaouna et al. / Biological Control 52 (2010) 37–45 41
insecticidal effect on CLM immature stages (Beattie et al., 1995;Rae et al., 1996a,b). Furthermore, the mean numbers of minesper leaf found during the 1997–1999 survey (1.8 to 2.4 vs. 3.2to 5.1 mines, respectively) were higher than those observed in
2006. Maximum values were also greater in 1997–1999 survey:up to 10 mines per leaf at Montcada in October 1998, comparedto the highest value of 5.0 mines per leaf recorded at L’Alcúdia inAugust 2006.
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Fig. 4. Incidence of predation, feeding punctures and parasitism on citrus leafminer immatures (percentage of susceptible leafminer stages) in three different Clemenulesclementine orchards (a, Bétera; b, L’Alcúdia; c, Llíria).
42 F. Karamaouna et al. / Biological Control 52 (2010) 37–45
The number of parasitoids per leaf recorded during 1997–1999ranged between 0.01 and 1.6 and these values are similar to those
recorded during 2006. However, parasitoid guild compositionchanged drastically and the introduced species C. phyllocnistoides
Predation
71.3%
15.5%
8.9%4.3%
LII LIII
LIV PupaParasitism
1.1%23.0%
66.5%
Feeding puctures
74.4%
17.0%
Predation
43.4%
1.0%
52.9%
2.6%
Parasitism
67.5%
31.5%
Feeding puctures
19.3%
80.4%
1997-1999
2006
Fig. 5. Percentage of CLM immature stages parasitized or preyed upon in 1997–1999 (data from Urbaneja et al. (2000)) and in 2006.
1997-1999
0102030405060708090
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Stage
Num
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LII LIII LIV Pupa Adults
LII LIII LIV Pupa AdultsStage
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Healthy ParasitizedPierced Predated
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Fig. 6. Stage specific overall natural enemy-caused mortality on CLM second instar to adulthood in 1997–1999 (a) (Urbaneja et al., 2000) and in 2006 (b).
F. Karamaouna et al. / Biological Control 52 (2010) 37–45 43
became dominant. As pointed out by other authors (Urbaneja et al.,2003b; Garcia-Mari et al., 2004) this parasitoid displaced most ofthe opportunistic indigenous parasitoids that had moved ontothe CLM upon its introduction in Spain. This situation is similarto one of the most cited examples of competitive displacement ofone species by another in biological control: the aphelinid idiob-oints Aphytis melinus DeBach and Aphytis lingnanensis Compereon A. aurantii in California (Briggs et al., 1999; Luck et al., 1999).A. lingnanensis is an exotic parasitoid that had been moderatelysuccessful at controlling A. aurantii, but it was rapidly displacedby another introduced species, A. melinus, in Southern California
in coincidence with higher suppression of A. aurantii. A. melinusoutcompeted A. lingnanensis because a female offspring could suc-cessfully develop on slightly smaller hosts. Similarly, C. phyllocnis-toides, which parasitizes and feeds preferentially on CLM secondinstars (Fig. 5; Urbaneja et al., 2003b), outcompeted native parasit-oids, which mainly utilize bigger CLM third instars (Urbaneja et al.,2000). Furthermore, establishment of C. phyllocnistoides modifiedthe relative abundance of the different stages of CLM in the field.Because C. phyllocnistoides is an idiobiont, CLM second instars be-came dominant in the orchards and subsequent stages became lessabundant. Accordingly, stage-specific natural enemy-caused
44 F. Karamaouna et al. / Biological Control 52 (2010) 37–45
mortality changed (Fig. 5). This shift in the relative abundance ofCLM immature stages in the field from third to smaller second in-stars could partly explain the decrease in the damage to citrus fo-liage observed by Garcia-Mari et al. (2004), but the secondaryeffects linked to this shift cannot be ignored. As expected, general-ist predators preyed mostly on CLM second instars instead of thirdinstars (Fig. 5). The enormous natural enemy-caused mortality onCLM second instars is likely the cause for the displacement of na-tive non-specific parasitoids, mainly parasitizing and feeding onthird instars (Urbaneja et al., 2000), from the system (Table 1).Therefore, a switch of the feeding preferences, which often occursin non-specific species in response to the relative availability ofalternative food sources (Murdoch, 1969; Murdoch and Oaten,1975), could explain the movement of native natural enemies ontothe CLM in the first place, as well as the later shift of predators toCLM second instars and the decrease of indigenous parasitoids onolder instars upon establishment of C. phyllocnistoides. The nativeparasitoid species have probably moved back to their original hosts(Costa-Comelles et al., 1997; Garcia-Marí et al., 1997), leaving CLMfourth instars and pupae almost free of parasitism (Fig. 6). P. pectin-icornis, for instance, is known to exhibit a wide host range ofmainly lepidopterous and coleopterous leafminers on deciduoustrees (Boucek and Askew, 1968), as well as to parasitize the olivefruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae) (Neu-enschwander et al., 1986). It is difficult to know about the nativeoriginal hosts of the other previously predominant eulophid, C. bre-vis, as it was first described subsequent to P. citrella introduction inSpain (Schauff et al., 1998; Zhu et al., 2002).
Parasitism rates given by Garcia-Mari et al. (2004) (60%) are muchhigher than those found in this study (18%). Methodological differ-ences could account for this discrepancy (Luck et al., 1988, 1999).Our results, though, roughly coincide with those produced by Xiaoet al. (2007) in citrus in Florida in a completely different environ-ment. These authors found an overall mortality of 89.0% from CLMfirst instar to adulthood (59.6% predation plus host feeding and29.8% parasitism). In our case, mortality from first instar to adult in2006 was 93.3% (18.0% parasitism, 40.8% feeding punctures and34.5% predation) (Fig. 6). However, contrary to what Xiao et al.(2007) found, ants are not the key predators in Spain, possibly be-cause different species were present (Urbaneja et al., 2004). Never-theless, the exact identity of CLM predators in Spain remains unclear.
In summary, our results provide evidence of increased naturalenemy-caused mortality of CLM in 2006 compared to 1997–1999in correspondence with decreased CLM incidence over the sameperiod. Mechanisms such as competitive displacement may haveoccurred and the new assemblage of natural enemies feeding onCLM has led to a better regulation of its populations.
Acknowledgments
P.A. Stansly (University of Florida) and E. Grafton-Cardwell(University of California) provided useful comments on an earlydraft of the manuscript. We are grateful to M. Llavador, S.L. MartíNavarro and V. Borràs for allowing us to sample in their orchards.F. Karamaouna was recipient of a grant from the Organization forEconomic Co-operation and Development (OECD). This work waspartly funded by Spanish Ministerio de Ciencia e Innovación(AGL2005-07155-C03/AGR and AGL2008-05287-C04/AGR), andthe Conselleria d’Agricultura, Pesca i Alimentació de la GeneralitatValenciana.
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