INITIAL STUDY OF REARING AND RELEASE OF Anthocoris minki Dohrn (HEMIPTERA: ANTHOCORIDAE) FOR BIOLOGICAL CONTROL OF Agonoscena pistaciae Burckhardt and Lauterer (HEMIPTERA: PSYLLIDAE) IN PISTACHIO ORCHARDS ABSTRACT The pistachio psyllid, Agonoscena pistaciae Burckhardt & Lauterer 1989 (Hemiptera: Psyllidae), is an important pest of pistachio in Şanlıurfa, Turkey. In this study, laboratory reared Anthocoris minki Dohrn (Hemiptera: Anthocoridae) was released for biological control of pistachio psylla in pistachio orchards. Frozen eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were utilized as prey of rearing of A. minki in the laboratory. The experiment was carried out between May and November in 2006. In the second week of July, A. minki caused reduction the density of pistachio psyllas’ from 175.6 to 31.3 nymph/composite leaf. However, in the beginning of August, due to infestations from a neighboring orchard, population density of the pistachio psylla

The pistachio psyllid, Agonoscena pistaciae Burckhardt & Lauterer 1989 (Hemiptera: Psyllidae), is an important pest of pistachio in Şanlıurfa, Turkey. In this study, laboratory reared Anthocoris minki Dohrn (Hemiptera: Anthocoridae) was released for biological control of pistachio psylla in pistachio orchards. Frozen eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were utilized as prey of rearing of A. minki in the laboratory. The experiment was carried out between May and November in 2006. In the second week of July, A. minki caused reduction the density of pistachio psyllas’ from 175.6 to 31.3 nymph/composite leaf. However, in the beginning of August, due to infestations from a neighboring orchard, population density of the pistachio psylla increased to 193.4 nymph/composite leaf. Therefore, in the second week of July, an additional release of A. minki was performed. Towards the end of August the pistachio psylla population density was reduced to the economic injury threshold level. In the control orchard, where no A. minki was released, pistachio psylla reached the density of 458 nymph/composite leaf in spite of insecticide use. The results of this initial study indicated that this predator species may be effective for biological control of A. pistaciae.


INTRODUCTION
Pistachio production is of great economic importance in the Southeastern Anatolian Region, the main pistachio plantation area of Turkey. The common pistachio psylla, Agonoscena pistaciae Burckhardt & Lauterer (Hemiptera: Psyllidae) is a major pest of pistachio trees, Pistacia vera L. in Turkey since late 1980's (Mart et al., 1995). Also, A. pistaciae is the most serious insect pest species of pistachio trees and causes considerable damage in Iran and Greece (Mehrnejad, 2001;Souliotes et al., 2002), neighboring countries of Turkey. Overwintered psyllid adults appear on pistachio trees in mid-March and females lay eggs in mid-April and produce 5-6 generations a year (Çelik, 1981). Psyllid nymphs and adults interfere with kernel development, resulting in bud drop and defoliation. Both adults and immature suck plant sap and produce white crystallized honeydew. The control of this pest is difficult with insecticides because of its tendency to develop resistance to insecticides (Mehrnejad, 2001).
Anthocorids have been widely used in biological control for over a century and are considered as important natural enemies of pest species such as psyllids, thrips, mites and aphids (Lattin, 1999). Anthocoris nemoralis (Fabricius) (Hemiptera: Anthocoridae) has been released to control pear psylla in pear orchards Rieux et al., 1994;Unruh and Higbee, 1994;Anonymous, 2015). A. nemoralis and parasitoid Psyllaephagus pistaciae Ferriere (Hymenoptera: Encyrtidae) were reported to be the most abundant natural enemies on pistachio trees in Makrakomi area of Central Greece (Souliotes et al., 2002). The predator, A. minki is known as one of the native bio-control agents of A. pistaciae in pistachio production areas in Turkey (Bolu et al., 1999;Çelik, 1981;Mart et al., 1995;Yanik et al., 2011a) and Iran (Pourali et al., 2010). A. minki is highly active from early April to late October on pistachio trees (Bolu et al., 1999).
The objective of this study was to evaluate effectiveness of A. minki as biological control agent of pistachio psylla. We believe this investigation is the first study on A. minki as a predator of the A. pistaciae. This study provides initial information for understanding the rearing and release of A. minki. Obtaining knowledge about this predator will help to reduce chemical applications against the pistachio psylla.

MATERIAL AND METHODS Predator rearing.
A. minki adults were collected from pistachio orchards in Sanlıurfa in 2005 and reared in the laboratory using predator A. nemoralis's rearing method as described by Samsoe-Petersen et al. (1989) and Yanık and Uğur (2005), and kept under 25±1°C, 60±10% RH and 16L8D photoperiod conditions. About 50-100 adults were placed in a plastic cup (500 ml). The lid of the cup contained a ventilation hole covered with a fine-mesh nylon screen. E. kuehniella eggs were supplied to the nymphs and adults as a nutrient source. Fresh bean pods (Phaseolus vulgaris L. (Fabaceae)) were added to each container as oviposition substrates. E. kuehniella eggs and fresh bean pods were replaced every two or three days. Fresh bean pods containing A. minki eggs were placed in a plastic container (250 ml) with a ventilation hole on its lid. E. kuehniella eggs were put near the fresh bean pods, food source for newly emerged nymphs. The diets and fresh bean pods were replaced every two or three days.
E. kuehniella was reared on a wheat and wheat-bran mixture at 2:1 ratio (Bulut and Kılınçer, 1987). The mixture was placed in plastic cups (27x37x7 cm) and E. kuehniella eggs were placed on the mixture for growth. Plastic cups were covered with a piece of gauze. After the emergence of E. kuehniella adults were transferred into a plastic cage (15x25x10 cm), having plastic screens on two opposite sides, for mating and oviposition. Rearing of E. kuehniella were done at 25±1°C and in constant darkness.
Field releases. A total of 5 500 A. minki adults were released into a 10 to 15 year old pistachio orchard of 220 trees in the area of Ogutcu, Sanliurfa (ca. 147 trees per ha, avg. height 2.55 m, tree canopy diameter 2.64 m, 6.4 extension shoot, n=10) on different dates, May 4 and 24 (7 adults per tree), June 22 (4 adults per tree) and July 12 (7 adults per tree). Age and sex ratio of released adults 3-7 day-old and 1:1, respectively. A total of 25 A. minki adults were released per tree. First release was performed when approximately 30-40% of the eggs from overwintered female psyllas were hatch. In the laboratory, adults of A. minki were put in a small plastic cups, sawdust were provided for hiding. In the field each cup was emptied onto branches of tree. Ten trees were assigned for treatment and marked. Leaf and beating samples were taken at ten day intervals from May to November to assess the effect of the releases. Since trees were young, only a total of 20 leaves were picked from the marked trees and sides of a tree were reversed each week to minimize arthropod distribution. Leaf samples were taken to the laboratory in cooling boxes and immediately checked for arthropods under a stereomicroscope. A beating sample consisted of dislodged arthropods from tree branches were taken. After counting the beating samples, arthropods were released to the orchards.
No insecticide was applied in the release orchard. However, a fungicide Dodine was applied to trees for Septoria pistaciae disease on May 20 and June 4, 2006. An orchard with 200 trees next to the release orchard was selected as a control orchard (Control-I). No release was made, but only fungicide (Dodine) was used in Control-I orchard. Another orchard with 120 trees and 150 m away from the release orchard was used as the second control (Control-II). In addition to the same fungicide, trees received two insecticide (Phosalone) applications against pistachio psylla in Control-II orchard on June 8 and July 28, 2006. Due to the considerable defoliation and honeydew production, counting of psylla nymphs and eggs was stopped in early August in Control-II orchard.
Determination of overwintering predator insects. Tree bands of corrugated cardboard were used to provide shelters (Civolani and Pasqualini, 2002;Horton and Lewis, 2000;Horton, 2004) for overwintering A. minki and other predator insects. Each band was 7 cm wide and long enough to encircle the trunks of pistachio trees. Corrugations were 4 X 5 mm, large enough to allow insects fit in. Bands were placed around the trunks, 0.3 m above the ground. Nine trees were banded with corrugated cardboard traps in the release orchard on November 9, 2006. The traps were collected on February 22, 2007 and placed in large plastic bags. Bags and bands were checked every 3 or 5 days for predators in the laboratory from late February to early July. Insects were aspirated from the plastic bags and counted.
The correlation between A. minki and A. pistaciae was evaluated using linear regression model.

RESULTS AND DISCUSSION
Field releases. The results indicated that there was a significant positive relationship between pistachio psylla and A. minki in the release orchard (F 2,157 =20,2659, P<0,01) ( Figure 1). Predator populations increased as the psylla population increased. Releases on May and June led the number of predator to increase by 14.93±3.97 adult + nymph/beating in the beginning of July and psylla population decreased from 175.6±50.38 to 31.3±13.75 nymph/composite leaves in the second week of July.
However, due to psylla infestation from an adjacent orchard (another side of release orchard, apart from Control-I orchard) o in the second week of July, psylla population increased up to 193.4±39.67 nymph/composite leaves in the release orchard in the first week of August. Therefore, predators were released for the fourth times in the release orchard on 12 July 2006. Psylla population was reduced to 25.1±7.07 nymph/composite leaves in the last week of August, after six weeks from the fourth release.
The highest number of psylla nymphs in Control-I was 539.40±91.57 nymph/composite leaves and occurred in the second week of October; while it was only 70.4±20.67 nymph/composite leaves in the release orchard.
Significant differences between release orchard and Control-II orchard in terms of psylla populations occurred one week after the second release. At the beginning of June, there was about 370.60±32.28 psylla nymphs/composite leaves in Control-II orchard.
Insecticide application reduced the number of psyllids to 52.0±15.17 nymph/ composite leaves in the second week of June. However, psyllid population increased at the end of July and reached 458.00±56.53 nymph/composite leaves and an additional insecticide application reduced the psyllid population to 135.83±23.96 nymph/composite leaves at the beginning of August.
Population of psylla did not decrease below economical threshold level in Control-II orchard. The highest number of A. minki was 0.44±0.12 adult + nymph/beating in Control-II orchard, which was considerably lower than those in the release orchard.
Other insect natural enemies. Total numbers and the occurrence rates of insect natural enemies of pistachio psyllid in the release and control orchards between May and November are shown in Table 1. A. minki was the most abundant species in the release orchard, followed by the nymph parasitoid Psyllaephagus sp. (Hym.: Encyrtidae) and Coccinellidae species. Psyllaephagus sp. was more abundant in the control orchards considering the other natural enemies. Compared to the release orchard, A. minki numbers were lower in Control-II orchard due to insecticide applications. Some of the natural enemies that were observed in the other orchards were not found in Control-II orchard.  Population changes of other relatively abundant predators (Psyllaephagus sp. and Oenopia (Synharmonia) conglobata (L.) (Coleoptera: Coccinellidae) in the orchards are shown in Figure 2. In the release orchard, population of parasitized nymphs by Psyllaephagus sp. increased when the population of A. minki decreased after mid July and in the beginning of September and decreased when population of A. minki increased in the beginning of August (Figure 1 and 2), indicating a negative relationship between the two natural enemy populations.
Total number of hatched and live eggs of A. minki per composite leaves is shown in Fig. 3. Number of live eggs reached the highest level (5.85±2.36) in July, and decreased starting from August and no live eggs were observed after the middle of September.

DISCUSSION
The most important predator of pear psylla, A. nemoralis, is mass reared by specialist companies and released for biological control of pear psylla in pear orchards Rieux et al., 1994;Sigsgaard et al., 2006a;Sigsgaard et al., 2006b;Unruh and Higbee, 1994;Anonymous, 2015). No study is available on biological control of pistachio psylla by A. minki. However, this predator species was successfully reared in the laboratory using the method to rear A. nemoralis (Samsoe-Petersen et al., 1989;Yanık and Uğur, 2005).
E. kuehniella eggs, commonly used to rear anthocorid species (Arijs and De Clercq, 2001;Bueno et al., 2006;Fauvel et al., 1994;Murai et al., 2001;Samsoe-Petersen et al., 1989;Yanik and Unlu, 2011;Yanik and Unlu, 2015), were used to rear A. minki. Since anthocorid bugs lay their eggs in the tissue of plants, faba beans have been commonly used as the oviposition substrate for Orius spp., the anthocorid predators (Castañé and Zalom, 1994;Linus et al., 2002;Murai et al., 2001;Ruberson et al., 1991;Richards and Schmidt, 1996;Venzon et al., 2002). Faba beans that can be obtained throughout the year, an advantage for mass rearing (Murai et al., 2001), were also successfully used as the oviposition substrate for A. minki. Anderson (1962) reported that A. minki prefers psyllids as prey. Al-Maroof (1990) stated that poplar psylla (Camarotoscena speciosa Flor (Hemiptera: Psyllidae)) was an important pest of the poplars and A. minki was the most common predator species and constituted 81% of the predators. Besides, A. minki was an important and effective predator of Pemphigid aphids that cause galls on Populus nigra and P. nigra var. italica in Europe (Foster, 1990;Urban, 2002;Urban, 2004). Yayla (1983Yayla ( , 1984 reported that A. nemoralis was the most effective predator of the olive cottony aphid (Euphyllura olivina (Costa), Hemiptera, Aphalaridae)), while A. minki was the second important predator. Clearly, A. minki feeds on psyllids and aphids and the results of this study indicated that it is capable of suppressing the pistachio psylla. Souliotes et al. (2002) indicated that A. nemoralis, Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) and endoparasite, P. pistaciae have an important role in biological control of A. pistaciae and there is a positive relationship between the population of A. nemoralis and A. pistaciae. The result of our study also revealed a positive relationship between A. minki and A. pistaciae in the pistachio orchards (Figure 1). The first release was performed on May 4 when 30-40% of the eggs from overwintering psyllids hatched. Second release increased the A. minki population above the naturally occurring level and kept the pest population at the economical threshold level in the second half of July (the economical threshold level; 25-30 nymph/composite leaf, Mart et al., (1995)). The populations of psylla nymphs were reduced by A. minki by 51% nine days after the second release and by 82% three weeks after the third release.
Although the Control I orchard was near the release orchard, psyllid population was rather low until September. According to long years of observations, psylla population ranges from orchard to orchard, and from tree to tree as well. Starting from September psylla population increased in Control I orchard. Early defoliation of pistachio trees caused by high psylla population brought about to drop fruit buds too, on which fruit of next year would develop.
Due to immigration of gravid adult psyllids from the unmanaged adjacent orchard, psylla population began to increase in release orchard after the second week of July. Because the release orchard was small, the immigration of adult psyllids from adjacent orchards onto the experimental plots was a risk. The fourth release decreased the pest population to 25.1 nymph/composite leaf level within six weeks, which is the lower limit of the economical threshold for this pest. Sisgaard et al. (2006aSisgaard et al. ( , 2006b showed that during the release of A. nemoralis and A. nemorum (Linnaeus) (Hemiptera: Anthocoridae) in small experimental fields, immigration of Cacopsylla pyri Linnaeus (Hemiptera: Psyllidae) from an adjacent orchard reduced the effectiveness of A. nemoralis. Authors suggested that more than one release was necessary to maintain predators at higher population levels in release orchards.
Although, the number of psylla nymphs decreased immediately after the insecticide applications in Control II, it increased rapidly afterwards (Figure 1). Due to the pesticide applications, the number of natural enemy species and their density remained lower in the Control-II than in the release and the Control-I orchards (Table 1). Despite the two insecticide applications, A. pistaciae population did not decrease to the economical threshold level in Control-II orchard.
Due to the releases, the number of A. minki was the highest in release orchards, whereas the number of parasitized nymphs by Psyllaephagus sp. was the highest in control orchards (Table 1). This negative relationship between A. minki and Psyllaephagus sp. could be due to the fact that A. minki feeds on parasitized nymphs by Psyllaephagus sp.. Parasitoids appeared after the first week of June and reached the highest population density in late September and early October (Figure 2) when the ratio of parasitized and survived nymphs of A. pistaciae is approximately 1:10. This not significant contributes to the limitation of the population of A. pistaciae. According to Souliotis et al. (2002), the ratio of parasitized with P. pistaciae and survived nymphs of A. pistaciae is approximately 1:1 and this ratio contributes to the restriction of the population of A. pistaciae at the end of September/beginning of October.
A. minki females lay their eggs in the tissues of plants such as leaves throughout the season. The average number of survived eggs per leaf was 5.85±2.36 in July (Figure 3). There were no survived eggs on leaves after the mid-September. This suggests that A. minki population may not increase even A. pistaciae population increases after the mid-September. The mean number of eggs laid end of the season was 48.55±11.97 eggs/composite leaves. Sisgaard et al.(2006b) reported that the theoretical egg number per tree was 11 A. nemoralis eggs which halved the C. pyri population compared to not release trees.
Overwintered predator species in an fruit orchard may be potential sources of biological control in orchards (Yanik et al., 2011b).
Establishment of released natural enemies in release area is important for the sustainability of biological control. In this case, the ability of biological control agent to overwinter within the orchard is a critical factor. A. minki was able to overwinter in the corrugated cardboard traps in the orchards, indicating that this species could survive on trees in winters.
As a result, one year release of A. minki against A. pistaciae led to effective control of this pest. Other predators can also contribute to decrease psyllid population. In this study O. conglobata and Psyllaephagus sp. may be as important as anthocorids in controlling psyllid. Overall, our findings may provide important background for biological control studies in pistachio orchards in the future, because this was the first rearing and release study using A. minki.