The aphidophagous predator Harmonia axyridis (Coleoptera: Coccinellidae) in Greece, 1994-1999

During the years 1994–1999, several hundreds of thousands of H. axyridis adults were released at various cultivations infested by aphids (citrus, vegetable and bean crops, maize etc.) and on ornamental plants in urban settings in central and southern Greece (mainly Attica and Peloponessos region) as well as on several islands. Between 1995–1999, spring sampling was conducted in some areas, just prior to releases, in order to determine if H. axyridis overwintered in the field. No presence of H. axyridis was recorded in any of the orchards where the predator had been released save in spring of 1998 and 1999 when small colonies (<50 individuals) of overwintered H. axyridis adults were observed in the Attica region. The above results suggest an inability of released H. axyridis populations to establish in Greece, although in some areas this species became an important biocontrol agent during the growing season.


INTRODUCTION
Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is a predator used as a biocontrol agent against a wide range of soft bodied insects (aphids and scales) (Katsoyannos, 1996, Pervez & Omkar, 2006).An expectation has recently been expressed that, because of some life-history parameters, this species may become a serious competitor of Coccinella septempunctata in particular habitats (Hodek & Michaud, 2008).However, its effectiveness as a biological control agent has been accompanied by an apparently adverse impact on other aphidophagous coccinellids (Adriaens et al., 2003, Koch & Galvan, 2008).In addition, Harmonia axyridis has caused problems in fruit production (Koch, 2003) and as a household nuisance as it can induce allergic rhino-conjunctivitis in some people (Koch et al., 2006, Majerus et al., 2006, Pervez & Omkar, 2006).
During spring 1994, approximately 620 adults of H. axyridis were released in four citrus growing areas of Greece (Marathon-Attica, Campos-Chios, Leonidion-Arcadia, Chania-Crete) on orange, mandarin and sour orange infested by aphids [Toxoptera aurantii, Aphis spiraecola and A. gossypii (Hemiptera: Aphididae)] ( Katsoyannos et al., 1997).Over the next two months, H. axyridis proved to be an effective biocontrol agent against the afore-mentioned aphids in two locations (Campos-Chios and Leonidion-Arcadia).In outdoor cages H. axyridis overwintered in the adult stage (~30% of the adults of the 4 th generation survived), and under conditions of a continuous surplus of aphids completed four overlapping generations annually and did not diapause during summer.In Greece, aphid populations are scarce in summer (Katsoyannos et al., 1997).
The purpose of the current study is to present the status of H. axyridis in Greece during the years 1995-1999, when increased numbers of the predator were released over more extensive areas.

Harmonia axyridis rearing and releases
After the first releases of H. axyridis in Attica, Chios, Arcadia and Crete in 1994 (Katsoyannos et al., 1997) the rearing of the predator and release programme was continued by the laboratory of Biological Control of Benaki Phytopathological Institute.H. axyridis was reared on Aphis fabae (Hemiptera: Aphididae) on Vicia faba as well as on Dysaphis crataegi (Hemiptera: Aphididae) on squash (Cucurbita pepo, C. maxima and C. moschata) in controlled conditions (temperature: 25 ± 1°C; relative humidity: 65 ± 2%; photoperiod: 16L : 8D).From 1995 to 1999 more than 100.000insectary-reared adults of H. axyridis were released in various cultivations (citrus, vegetables, beans, maize) infested by aphids, or in urban areas on ornamental plants, in central and southern Greece (mainly Attica and Peloponessos) and the islands of Chios, Euvoia and Crete (Table 1).Moreover, during the years 1997 through 2002, several hundreds of thousands of imported H. axyridis individuals were released by private companies mainly in urban areas of the mainland (Attica, Peloponessos) and on the islands of Corfu, Rhodos and Crete.Afterwards, releases of the predator were terminated due to the high market price of the predator and not due to legal prohibitions.

Sampling method
During 1995-1999, samplings were conducted every spring just before releases were made, in thirteen areas (I, II, III, IV, VII, XI, XV, XVII, XVIII, XIX, XX, XXII, and XXVI) in order to determine if any H. axyridis had overwintered in the field.A second sampling was conducted a month later in order to estimate the population size of the exotic predator shortly after the release.Twelve samples were collected from each location on each sampling date.In arboric cultivations and ornamental trees, branch beating over a 1 m 2 canvas area was applied, whereas in other cultivations (vegetables, beans, maize, ornamental shrubs) a 20 cm-length shoot of the plant was used as a sampling unit.* Release-locations of H. axyridis adults in 1994 (Katsoyannos et al., 1997).

RESULTS AND DISCUSSION
A month after the 1994 releases, H. axyridis became an important biocontrol agent at the Campos-Chios and the Leonidion-Arcadia locations (Katsoyannos et al., 1997).In spring 1995, however, the presence of H. axyridis was not recorded in any orchards.
In the following years, despite continued massive releases of H. axyridis, only in spring 1998 and spring 1999 were small colonies (< 50 individuals) of overwintered H. axyridis adults found in the Attica region, although in some of the locations the released predator became abundant during the season (Table 1).The above result shows that although H. axyridis has some ability to overwinter in Greece, this has occurred only rarely and in very low numbers of adults.
Although the ecoclimatic indices for Greece have been predicted by Poutsma et al. (2008) as appropriate for H. axyridis establishment, the predator seems so far to have failed to establish despite continued releases.However, absence of evidence does not necessarily mean evidence of absence.In regions of the Western Hemisphere (USA, Mexico) where the climatic conditions are similar to those in Greece, H. axyridis was not established during the intentional releases but later in time and in a long distance from any of the initial release sites (Koch et al., 2006;Koch & Galvan, 2008).
It is noticeable that in Greece the overwintering H. axyridis adults were observed only at the location of Varympompi Attica, the only area where the exotic predator was released in Vicia faba fields infested by A. fabae, the prey that was also used for the laboratory rearing.In contrast, in the other places (almost all the field releases of H. axyridis occurred in citrus cultivations where Toxoptera aurantii, Aphis spiraecola and A. gossypii were the aphid pests) no overwintering H. axyridis individuals were recorded.However given the known polyphagous habits of the predator (Lucas et al., 1997(Lucas et al., , 2002;;Michaud, 2001Michaud, , 2002Michaud, , 2004) ) as well as the evidence from our study that the predator became an important biocontrol agent during the season, the failure of the establishment of the exotic predator could not be attributed to prey identity.
A similar case of a failure in establishment of H. axyridis was observed by Soares et al. (2008) at Santa Maria of the Azores islands where more than one hundred thousand third instar larvae were released into several citrus orchards.Although after the releases there were records of individuals that dispersed and fed on citrus, apple trees and bean plants, the exotic predator failed to establish (Soares et al., 2008).According to this study, the released populations failed to establish due to ecological factors such as the maladaptation to the local conditions and functional diversity saturation.It is likely that these factors also apply for the failure of H. axyridis' establishment in Greece.
In any case, given the current adverse impact of H. axyridis in other European countries and North-America, (Koch, 2003;Koch et al., 2006;Roy et al., 2006;Babendreier, 2007;Eschen et al., 2007;Brown et al., 2008a, b;Koch & Galvan, 2008) it would not be wise to continue field releases of this insect.This has been recently concluded by scientists working on risk assessment of biological control agents (van Lenteren et al., 2008;Roy & Wajnberg, 2008).Finally, scientists should be alerted as H. axyridis has shown a lag phase in its establishment (Soares et al., 2008) and in the future H. axyridis might resurface elsewhere in Greece.

TABLE 1 .
Releases and records of H. axyridis during 1995-1999 in Greece.