Displacement of Aphytis chrysomphali by Aphytis melinus , parasitoids of the California red scale , in the Iberian Peninsula

Parasitoids are the main natural enemies of the California red scale, Aonidiella aurantii (Maskell) and on occasion can regulate their populations. To increase their effectiveness, inoculative or augmentative releases of parasitoids are promoted. Previous to the implementation of any release strategy an important and necessary step is to acquire knowledge on the parasitoid fauna associated with this key phytophagous pest. Parasitoids were surveyed and quantified in Spanish citrus orchards between 2005 and 2009. Aphytis melinus DeBach (87.1%) resulted as the dominant species, followed by Aphytis chrysomphali (Mercet) (15.9%), Encarsia perniciosi (Tower) (2.4%) and Aphycus hederaceus (Westwood) (0.004%). Overall, higher levels of parasitism were recorded in fruit than in twigs. Scales in fruit were parasitized at similar levels by the different parasitoid species whereas E. perniciosi was more active in twigs. Data eventually reveal the recent displacement of A. chrysomphali by A. melinus. The implications of these results on the biological control of A. aurantii are discussed and this information will be useful in the decision of IPM strategies for this pest. Additional key words: biological control; Aonidiella aurantii; climatic conditions; Encarsia perniciosi. * Corresponding author: pilarvanaclocha@gmail.com Received: 08-11-13. Accepted: 20-02-14. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(1): 244-251 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2014121-5266 eISSN: 2171-9292


Introduction
Hymenopteran parasitoids of the genus Aphelinidae are the main natural enemies of armoured scale insects, particularly the ectoparasitoids of the genus Aphytis (Rosen, 1994) and the endoparasitoids of the genus Encarsia (Viggiani, 1990).Several species of these genera have been widely used in the regulation of California red scale (CRS), Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae) populations (Viggiani, 1990;Rosen, 1994).This phytophagous pest, CRS, can cause the commercial depreciation of fruit and due to its unsatisfactory control is considered key pest in almost all citrus-growing areas worldwide (Jacas & Urbaneja, 2010).
Natural occurring CRS parasitoid guilds are insufficient to keep pest populations under economic injury levels (Viggiani, 1994).Hence, to increase the benefits provided by these natural enemies, biological control (BC) programs, based on inoculative or augmen-tative releases of CRS parasitoids as well as conservation measures addressed to maintain naturally occurring parasitoid guilds, are being promoted (Jacas & Urbaneja, 2010;Tena et al., 2013b;Vanaclocha et al., 2013a;Urbaneja et al., 2014).Previous to the implementation of any of these measures an important and necessary step is to acquire knowledge on the parasitoid fauna associated with this phytophage, such as guild composition, ecology and activity in the areas where they are going to be implemented.Previous studies have been conducted on A. aurantii parasitoids in Spanish citrus orchards (Rodrigo et al., 1996;Pina et al., 2003;Sorribas et al., 2008).Unfortunately, all of them were performed in the same citrus growing area, the Valencia region.In these studies, the parasitoids associated with A. aurantii were the ectoparasitoids Aphytis chrysomphali (Mercet), Aphytis melinus De-Bach, Aphytis lingnanensis Compere, Aphytis hispanicus (Mercet) and the endoparasitoid Encarsia perniciosi Tower.Aphytis chrysomphali and A. hispanicus are considered native species in Spain.Aphytis chrysomphali was found to be the prevalent parasitoid in earlier studies (Rodrigo et al., 1996;Pina et al., Displacement of Aphytis chrysomphali by Aphytis melinus, parasitoids of the California red scale, in the Iberian Peninsula 2003), but at present, A. melinus has displaced the native one becoming the most abundant species associated with CRS (Vanaclocha et al., 2009;Sorribas et al., 2010;Tena et al., 2013a).Aphytis melinus was first introduced in the Valencia region in 1976 (Meliá & Blasco, 1980).Aphytis lingnanensis and E. perniciosi were also introduced in this region, but their establishment was not successful (Rodrigo et al., 1996;Pina et al., 2003;Sorribas et al., 2010).
Parasitoids of CRS are usually found as species guilds that may vary in their composition at different sites and different substrates of the plant due to different environmental condition requirements as well as host stage preferences (Yu et al., 1990;Rodrigo et al., 1996).For their ecological success, parasitoids must adapt to a range of environmental conditions similar to the ones required by their host.However, when two or more species compete in the same ecological niche, small variations on any environmental variable may change the dominance hierarchy within the guild (Chapin et al., 2000;Hance et al., 2007).As a result, a reduction in the relative abundance of some species or even their extinction could result (Hance et al., 2007).The effect of environmental variables on parasitoid guilds can be followed through geographical or temporal studies.Areas under different climatic conditions may present different guild compositions.Temporal series in which these variables change may explain the ecological succession from a past to a present guild.Thus, climate change is recognised as an important factor affecting the distribution of a wide range of organisms (Pimm, 2001;Parmesan, 2006;Hance et al., 2007).Meta-analysis estimates an average displacement per decade of northern and altitudinal species distribution boundaries of 6.1 km and 6.1 m northward and upward respectively (Parmesan & Yohe, 2003).In addition to the more predictable effects of abiotic factors on single species, species idiosyncratic phenological responses to changes in environmental variables are expected to trigger a cascade of changes on species interactions at all ecological levels with unpredictable consequences (Davis et al., 1998;Chapin et al., 2000;Van Nouhuys & Lei, 2004).In the present work, a comprehensive study describing CRS parasitoid assemblages in the main Spanish citrus growing areas has been conducted.The species composition and their relative abundance have been determined for each assemblage and correlated to the CRS parasitism ratios found.Results obtained were compared with those of a previous study done on a more local scale (Sorribas et al., 2010).The information compiled provides a better understanding of the mechanisms determining the CRS parasitoid hierarchy under most of the climatic conditions found on the Iberian Peninsula, and help to predict potential changes in the hierarchy with regard to a global warming scenario.The information herein provided may be used in the design of Integrated Pest Management (IPM) strategies adapted to more local conditions.

Study sites and sampling methods
Aonidiella aurantii-infested fruit and twigs of citrus trees (sweet orange and clementine mandarin) were sampled between 2005 and 2009, from August to November, in 21 orchards located throughout the main citrus-growing areas of Spain (Table 1 and Fig. 1).During this period, twig and fruit samples were randomly collected from each plot at least once per month and taken to the laboratory, where leaves were removed.The first generation of the scale (May-June) was not sampled since fruit was not available for parasitism comparison with twigs.Twigs of approximately 20 cm were examined under a binocular microscope.From each sample, both twig and fruit, irrespective of the number of scales present, up to ten individuals were checked to standardise the sampling.One hundred susceptible scales of A. aurantii per sampling site and date for parasitism (second nymphal stage, young females and prepupa and pupa males), were checked.Parasitoid species were determined according to their pupa pigmentation pattern and, when necessary, pupae were transferred into gelatine capsules for further development.Adults emerged from pupae were determined to a species level.

Data analysis
Active parasitism rate, given as an average ± standard error, was calculated as the number of live parasitized CRS over the total number of scales susceptible to being parasitized.According to the parasitoids obtained, percentages of active parasitism were classified in four different assemblage combinations found in the sampled orchards: 1) orchards where A. melinus was predominant (> 95% of the parasitoids found) (Am); 2) orchards where E. perniciosi was present (< 95% of the parasitoids were A. melinus and 5-100% were E. perniciosi) (Am + Ep); 3) coexistence of A. melinus and A. chrysomphali, but predominance of A. melinus (51-95% A. melinus and 5-49% A. chrysomphali) (Am + Ac) and 4) coexistence of A. melinus and A. chrysomphali but predominance of A. chrysomphali (≤ 50% A. melinus and ≥ 50% A. chrysomphali) (Ac + Am).Parasitism percentages were subjected to a two-way analysis of variance (factors: substrate and parasitoid assemblage) and the Bonferroni-test was used for mean separation at p < 0.05.Data underwent angular transformation before the analysis to meet the assumption of normality and homogeneity of variance.
Aphytis melinus was the most widespread and the predominant species in the majority of the study locations accounting for 81.7% of A. aurantii parasitism.
Aphytis chrysomphali and E. perniciosi accounted for 15.9% and 2.4% of CRS parasitism (Table 1).The incidence of A. hederaceus was negligible, with only three specimens found in the south of Spain in 2005.Aphytis melinus was present throughout all the study area, while A. chrysomphali and E. perniciosi had more restricted and rather allopatric distributions.Aphytis chrysomphali spread in the eastern citrus areas situated at around 37º 50' N latitude (locations 1 to 11 in Table 1 and Fig. 1); conversely, E. perniciosi was distributed south of that latitude, in the Andalusia region (locations 12 to 21 in Table 1 and Fig. 1).In the sympatric area encompassing A. melinus and A. chrysomphali (Valencia region and Balearic Islands), the incidence of the latter dropped from 55.5% in 2005 to 8.7% in 2009.In its distribution area, E. perniciosi reached on average, a relative abundance of 6.9%.No association between parasitoid relative abundances and citrus species was observed (Table 1).

Discussion
Aphytis melinus and A. chrysomphali are widespread biological agents of CRS worldwide (Dahms & Smith, 1994;Viggiani, 1994;Noyes, 2003).In Spain A. melinus was first released in the Valencia region in 1976 as part of a BC program; later it was also released in Andalusia in 1987 (Pina & Verdú, 2007a).According to our results, A. melinus has been established very successfully in all citrus-growing areas of Spain.Aphytis chrysomphali is considered an indigenous species.It was first found in Spain on Chrysomphalus dictyospermi (Morgan) (Hemiptera: Diaspididae) in Andalusia, the Valencia region and the Balearic Islands by García-Mercet (1912).Later, this ectoparasitoid was found on both C. dictyospermi and CRS in Andalusia (García-Mercet, 1930) and the Valencia region (Limón et al., 1976;Rodrigo et al., 1996).The results here obtained demonstrate that A. melinus has become the dominant CRS parasitoid species in Spanish citrus and seems to be progressively displacing A. chrysomphali, at least in the hottest and driest locations as suggested by Sorribas et al. (2010) in Valencian citrus.This displacement has also been observed in other Mediterranean regions (Siscaro et al., 1999).
Aphytis chrysomphali was the dominant species parasitizing CRS in Spain before A. melinus releases started.In Andalusia, there is no data about the time in which the displacement by A. melinus occurred, but it probably happened after the releases accomplished in 1987 (Pina & Verdú, 2007a).In our study, no A. chrysomphali specimens were found in that citrus growing area during the three seasons when samples were collected.In relation to the Valencia region, in the 90's, Rodrigo et al. (1996) found that A. chrysomphali accounted for 98% of CRS parasitisim.At the end of that decade the relative abundance of this parasitoid declined to 77.7% (Pina et al., 2003), and later, it dropped to 50% (Sorribas et al., 2008).The data presented in this work confirms this tendency showing a decline from 55.5% in 2005 to 8.7% in 2009.A similar parasitoid displacement of A. chrysomphali by A. lingnanensis (DeBach & Sisojevic, 1960) and later of A. lingnanensis by A. melinus (DeBach et al., 1969) occurred in California; in Greece, a displacement of A. chrysomphali by A. melinus also took place (De-Bach & Argyriou, 1967).
The very recent displacement of A. chrysomphali by A. melinus in Spain caused either by climatic conditions, persistent use of pesticides, competition with A. melinus or possibly a combination of all these factors, implies a change in the performance of A. chrysomphali in controlling CRS.The average high temperatures of the Spanish Mediterranean climate is favourable to the presence of A. melinus (Samways, 1985) while, A. chrysomphali and E. perniciosi are better adapted to more moderate climatic conditions (Abdelrahman, 1974;Pina & Verdú, 2007b).Indeed, the rapid displacement which occurred in Andalusia was probably partly due to the significantly warmer temperatures of this area in comparison to the east coast (Fig. 1).Sorribas et al. (2010) observed this phenomenon on a smaller scale in the Valencia region, where A. melinus was super-dominant in those assemblages found in warmer areas whereas A. chrysomphali was able to coexist via temporal niche partitioning in those areas with colder springs and winters.
As yet, we cannot explain why, according to the current data, A. chrysomphali and E. perniciosi spread in allopatric ranges, the former in northern regions and the latter in southern ones.
The higher percentages of parasitism in fruit (21.2%) than in twigs (15.7%) found in this work are similar to those found in other related studies (Atkinson, 1977;Asplanato & García-Marí, 2002).We have shown that A. melinus and E. perniciosi are sympatric in groves of southern Spain, and that the latter shows a preference for twigs.This coincides with the situation on the California coasts described by Yu et al. (1990), who stated that this resource partitioning could explain the coexistence of A. melinus and E. perniciosi.In this work CRS sampling was not conducted during the first CRS generation, May-June, when fruit is not present yet and therefore, A. melinus and E. perniciosi are forced to compete for the same resource.Future studies which focus on this period of year will help to discern the role played by E. perniciosi on CRS suppression and the importance of competition between these two parasitoid species in warmer areas.
Several authors established that parasitism rates around 20% could control CRS (DeBach et al., 1969).This percentage was reached in some of the orchards sampled in our study; however, unfortunately, parasitoids were unable to keep CRS populations under economic injury levels.The insufficient natural BC provided by CRS natural enemies, and especially by parasitoids, places CRS in the category of key pest in Spain (Jacas & Urbaneja, 2010;Urbaneja et al., 2014).In recent years, emphasis has been placed on implementing environmentally safe measures to control A. aurantii in Spain, rather than using traditional chemical insecticides (Vacas et al., 2012;Vanaclocha et al., 2012Vanaclocha et al., , 2013b)).For this purpose, augmentative releases of CRS parasitoids should be one of the alternatives considered for controlling this pest.According to our results, the recommended species to use in such augmentative programs is A. melinus because it is clearly well-adapted to Spanish citrus growing conditions.Furthermore, because E. perniciosi plays a role in controlling CRS in twigs, releases in those areas where it can become established should be continued.