Influence of the eggs of Ephestia kuehniella (Lepidoptera: Pyralidae) reared on different diets on the performance of the predatory bug Orius laevigatus (Hemiptera: Anthocoridae)

The predatory bug, Orius laevigatus (Fieber, 1860) (Hemiptera: Anthocoridae), is an important biological control agent and widely used for augmentative biological control of the western fl ower thrips, Frankliniella occidentalis Pergande, 1895 (Thysanoptera: Thripidae) in greenhouse crops. This bug is generally reared using the Mediterranean fl our moth, Ephestia kuehniella Zeller, 1879 (Lepidoptera: Pyralidae). The aim of this study was to determine the effects of E. kuehniella eggs produced by adult moths reared on different larval diets on the performance of O. laevigatus. The diets were 95% cornmeal + 5% yeast (CY diet), 53.3% cornmeal + 26.7% wheat bran + 15% Glycerine + 5% yeast (CBGY diet) and 53.3% wheat fl our + 26.7% wheat bran + 15% Glycerine + 5% yeast (WBGY diet). Laboratory studies started with newly emerged nymphs of O. laevigatus kept in a climate chamber at 27°C, 60% relative humidity and a 14L :10D photoperiod. Eggs of E. kuheniella adults that were fed on one of the three different larval diets were provided to the nymphs as a source of food and bean pods of water. Following adult emergence, all individuals were sexed, and female and male pairs were put in plastic containers (30 cc) with eggs of E. kuehniella and bean pods. Nymphal development time, fecundity and longevity of O. laevigatus were not differently affected by feeding on the eggs of E. kuehniella reared on the three diets. In addition, the daily and total egg consumption of adults of O. laevigatus were similar. Because the CY diet eggs contain more nutrients, their consumption by nymphs was lower. This study indicates that the CY diet is an adequate diet for rearing E. kuehniella for producing eggs for rearing O. laevigatus.


ORIGINAL ARTICLE
frozen and kept for rearing O. laevigatus. All diets used in the experiments in the current study were used in a previous study by Kurtuluş et al. (2020) on the most promising ways of rearing E. kuehniella in the laboratory.

Orius laevigatus
O. laevigatus were collected from pepper plants growing in open fi elds in Adana, Turkey in 2018 and maintained in the laboratory. The predatory bug was identifi ed by using the identifi cation key of Péricart (1972) and then reared separately in plastic jars (1 liter in volume) with a perforated lid (5 cm diameter). The jars were sealed with mesh cloth for ventilation and to prevent escape. Adults and nymphs of this predatory bug were fed (ad libitum) with U.V sterilized E. kuehniella eggs and provided with bean pods as an oviposition substrate and source of water. Adults and nymphs of this predatory bug were kept in separate cages to reduce cannibalism. Moreover, to avoid genetic degeneration, new individuals were collected in the fi eld and added to the laboratory colony at least twice a year.

Effects of the different artifi cial diests fed to Ephestia kuehniella on biological parameters of Orius laevigatus
These experiments started with 90 newly emerged nymphs of O. laevigatus kept in a climate chamber at 27 ± 1°C, 60 ± 10% relative humidity and a 14L : 10D photoperiod. Eggs of E. kuehniella fed on one of the three different diets were provided as a source of food and bean pods as a source of water for the nymphs. Three times a week, the nymphs were fed 75 min U.V-sterilized (UVB light 280-315 nm) E. kuehniella eggs glued to pieces of cardboard with Arabic gum. The nymphs were checked twice a day and nymphal development times were determined. Following adult emergence, all individuals were sexed, and one female and one male were put in a plastic container (30 cc) along with eggs of E. kuehniella and bean pods and the pre-oviposition, oviposition, post-oviposition, longevity and fecundity of females recorded under the same experimental conditions as above. The containers were checked daily until the end of the pre-oviposition period and then every two days to determine the other biological parameters of O. laevigatus. The experiments were terminated when all individuals were dead.

Consumption by Orius laevigatus of the eggs of Ephestia kuehniella reared on different artifi cial diets
This experiment started in each case with 45 newly emerged nymphs of O. laevigatus that were fed eggs of E. kuehniella produced in one of the three diets and provided with fresh bean pods as a source of water. The number of eggs of E. kuehniella consumed by the nymphs of O. laevigatus were determined every two days. Following adult emergence, all individuals were sexed and one female and one male were put in a plastic container (30 cc) for mating. After four hours, the males were transferred to other containers. The consumption of E. kuehniella eggs by O. laevigatus adults (females and males) was recorded over a period of 15 days. Two hundred fresh eggs were placed in each container every two days for both nymphs and adults.

Statistical analysis
The experiments were carried out in a completely randomized design. Kolmogorov-Smirnov test and Levene's test were used to determine normality and homogeneity of variance, respectively. One-way Analysis of Variance (ANOVA) of nymphal development time, pre-oviposition, oviposition, post-oviposition, longevity and fecundity of adults of O. laevigatus was carried out using Tukey's HSD post hoc mean separation. The nymphal development experiments were carried out in a completely randomized design with at least 30 replicates for each diet. Data on consump-generations of Orius spp. results in a reduction in the fi tness of these predatory bugs, which affects their effectiveness as biological control agents (Arijs & De Clerq, 2004;Ferkovich et al., 2007;Venkatesan et al., 2008;Bonte & De Clerq, 2008, 2010a. Inspite of this, lepidopteran eggs have been used as the standard food in the commercial massrearing of Orius spp. since the early 1990s (Alauzet et al., 1994;van Lenteren & Tommasini, 2003). Moreover, there is no evidence that the performance of predatory bugs used in biological control programs has been affected by their rearing diet (Bonte & De Clerq, 2010b). Bonte & De Clercq (2008, 2010a report that the reproductive fi tness of O. laevigatus is greater when reared on E. kuehniella eggs than on other artifi cial diets. Many studies have shown that E. kuehniella is a nutritionally superior food for natural enemies Specty et al., 2003;De Clercq et al., 2005b;Song et al., 2019;Ricupero et al., 2020). However, there are virtually no studies on the effects of different artifi cial diets on the nutritional quality of E. kuehniella eggs as a food source for species of Orius. Therefore, determining the effects of E. kuehniella eggs, which are obtained by rearing this moth on different larval diets, on the fi tness of O. laevigatus is important for optimizing the mass rearing of this predator specifi cally for biological control purposes. Therefore, the nutritional quality of E. kuehniella eggs in terms of the biological parameters and consumption by O. laevigatus were determined in this study.

Insect rearing Ephestia kuehniella
The initial colony of the Mediterranean fl our moth, E. kuehniella was obtained from the Biyolojik Tarım Company, Erzin, Hatay, Turkey. The moths were maintained for six months in the Laboratory of Entomology (hereafter referred as the laboratory), Department of Plant Protection, Çukurova University, Adana, Turkey before they were used in the experiments. Three different diets consisting, respectively, of 95% cornmeal + 5% yeast (CY diet), 53.3% cornmeal + 26.7% wheat bran + 15% Glycerine + 5% yeast (CBGY diet) or 53.3% wheat fl our + 26.7% wheat bran + 15% Glycerine + 5% yeast (WBGY diet) were used to rear the larvae of E. kuehniella in a climate chamber at 25 ± 1°C, 60 ± 10% relative humidity and a 14L : 10D photoperiod. One kg amounts of the three diets were prepared and each placed in a plastic container (34 × 23 × 7 cm) to which 10 eggs were added per 1 g of food. The CY diet (I) consisted of 950 g cornmeal and 50 g yeast; the CBGY diet (II): 533 g cornmeal, 267 g wheat bran, 150 ml glycerine and 50 g yeast and WBGY diet (III): 533 g wheat fl our, 267 g wheat bran, 150 ml glycerine and 50 g yeast. In each case the dry components were mixed fi rst and then glycerine was added to the mixture. Corrugated cardboard plates were placed inside the containers to provide a pupation site and then containers were covered with fi ne muslin cloth. Each moth colony was reared on one of the diets for one generation before the trials. Following adult emergence, the containers were checked three times a week when any adults of E. kuehniella were collected with the help of an electric suction aspirator and transferred to egg laying containers (1 liter in volume). Eggs were collected from these containers every two days. Some of these eggs were used for maintaining the E. kuehniella colony, while others were tion (nymphs and adults) were also analyzed using one-way ANOVA followed by Tukey's post hoc test. The consumption experiments were carried out in a completely randomized design with at least 15 replicates for each diet. All analyses were done using SPSS © (ver. 25).

Effects of the different artifi cial diets on the nymphal development of Orius laevigatus
In this study, the development time of second nymphal stage was shorter when the eggs produced by moths reared on the CBGY diet were provided as food. But the development time of the other nymphal stages and total development time of O. laevigatus were not signifi cantly affected differently by the eggs of E. kuehniella reared on the different larval diets (F 2,85 = 1.453, p = 0.240; Table 1).

Effects of different artifi cial diets fed to the moths on the consumption of their eggs by Orius laevigatus
The consumption of eggs by the different stages of O. laevigatus are recorded in Table 3. The consumption of eggs by nymphs of O. laevigatus differed signifi cantly on the eggs of the moths reared on the different artifi cial diets (F 2,42 = 5.845, p = 0.006). The nymphs consumed fewer of the eggs produced by moths reared on the CY diet. However, the different artifi cial diets used for rearing the moths did not signifi cantly affect the daily and total consumption of the eggs of these moths by adults of O. laevigatus in the fi rst fi fteen days of their adult life (F 2,42 = 0.155, p = 0.857).

DISCUSSION
The success in rearing anthocorids depends on several factors, with the type of food being one of the most crucial  * Means with same letter in the same column are not statistically signifi cant according to Tukey's HSD test (P < 0.05); ** Diet names were constructed using the initials of the ingredients in each diet. C -cornmeal; W -wheat fl our; B -wheat bran; G -glycerine; Y -yeast.   2010a) and 13.5 days on bean pods (Bonte & De Clercq, 2011). At 25°C and 26°C, total nymphal development is 13.2 days on geranium leaves (Pelargonium peltatum L.) (Alauzet et al.,1994) and 11.8 days on bean pods (Tommasini et al., 2004), respectively. In this study, total nymphal development time was shorter than that recorded in previous studies. This could be due to the different oviposition substrates used and that the temperature was higher in this study. Moreover, in the current study, the artifi cial diets did not signifi cantly affect the pre-oviposition periods. Cocuzza et al. (1997)  Anthocoridae)] range between 3.2 to 6.7 days on bean pods at 26°C. In the current study, the pre-oviposition period is similar to that reported in previous studies even though bean pods were provided as a source of water and an oviposition substrate. Longevity is another factor that can directly affect the fecundity of insects (Leather, 1988). Cocuzza et al. (1997) and Bonte & De Clerq (2008) report that the longevity of females of O. laevigatus is 41.9 and 50.2 days, respectively. However, Alauzet et al. (1994), Tavella et al. (1994 and Tommasini et al. (2004) report shorter longevities (34.5, 23.0 and 38.6 days, respectively) when reared under similar conditions. In the current study, longevity ranged between 24.83 to 26.44 days, which is lower than the average of the values reported in the previous studies. This could be due to the higher temperature used in this study. Zhang et al. (2012) and Ali et al. (2020) report that longevity decreases with increase in temperature when adults of Orius similis Zheng (Hemiptera: Anthocoridae) and Orius strigicollis (Poppius) (Hemiptera: Anthocoridae) are fed on Tetranychus cinnabarinus (Boisd.) (Acari: Tetranychidae) and Pectinophora gossypiella Saunders (Lepidoptera: Gelechiidae) eggs, respectively.
Because of their high nitrogen content, lepidopteran eggs are of higher nutritional quality than other food sources, including the predators' natural prey (Ferkovich et al., 2007). The developmental and reproductive fi tness of O. laevigatus is greater when reared on eggs of E. kuehniella Tommasini et al., 2004;Bonte & De Clerq, 2008). Fauvel et al. (1987) and Cocuzza et al. (1997), respectively, report that Macrolophus caliginosus Wagner (Heteroptera: Miridae) and O. laevigatus perform better when reared on eggs of E. kuehniella than on some of their natural prey. Aragón-Sánchez et al. (2018) report that the increase in a population of O. laevigatus fed on Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) eggs is similar to that when naturally feeding on E. kuehniella and F. occidentalis. Furthermore their fi ndings highlight the promising role of the eggs of S. exigua as an alternative food for use in laboratory tests. However, the cost of mass-rearing S. exigua is considerably greater than that of E. kuehniella. Moreover, Ferkovich & Shapiro (2005) note the presence of a specifi c nutritional factor, in certain lepidopteran eggs, enhances the fecundity of predators. Lifetime fecundity is often considered to be a crucial factor determining the fi tness of insects reared on artifi cial diets (Grenier & De Clercq, 2003). In the present study, the fecundity of O. laevigatus did not differ on the eggs of the moths reared on the three different artifi cial diets (Fig. 1). Tavella et al. (1994) and Tommasini et al. (2004) report that the fecundity of O. laevigatus is 104.6 and 118.6 eggs/ female, respectively when reared on eggs of E. kuehniella eggs. Other researchers report higher fecundities ranging from 141 eggs to 198.0 eggs/female (Alauzet et al., 1994;Cocuzza et al., 1997;Vacante et al., 1997;Bonte & De Clerq, 2008). In the current study, the average fecundity of O. laevigatus was 150 eggs/female. These differences may be due to the different temperatures and oviposition substrates used in the experiments.
The nymphs of this predator consumed fewer of the eggs produced by the moths reared on the CY diet (Table 3). Van den Meiracker (1999) Tommasini et al. (2004). This may be related to the nutritional value of the E. kuehniella eggs. Moreover, it is not possible to compare this value because the ingredients of the E. kuehniella diets used in the other studies is unknown. However, the artifi cial diets did not affect the daily and total consumption by adults of O. laevigatus in the fi rst 15 days of their life (Table 3). Van den Meiracker (1999) and Calixto et al. (2013) (2008) report that adult diet of O. laevigatus had a greater effect than the nymphal diet on its fecundity, which is directly related to their consumption. Moreover, adults of O. laevigatus fed on eggs of E. kuehniella were clearly able to compensate for any defi ciencies in their nymphal diet.
Understanding the role of artifi cial diets in the development, fecundity and egg consumption of predatory bugs could improve the success of biological control programs by ensuring a greater effi ciency of the predators used in augmentative releases. The use of the eggs of E. kuehniella as the main source of food in the mass rearing of O. laevigatus can increase production costs (De Clercq et al., 2005a). Based on the current study, the three artifi cial diets fed to the moth did not affect either the development or fecundity of O. laevigatus. In addition, the CY diet resulted in nymphs consuming fewer eggs but had no effect on the egg consumption of adults. Moreover, the CY diet has the advantage that it is cheaper, more easily produced and less likely to be contaminated due to the lack of liquid in the diet. For this reason, the CY diet could be a suitable standard diet for the rearing of O. laevigatus. Further research on the effects of the CY diet on some biological parameters of this bug in subsequent generations should be conducted. In addition, the effi cacy of parasitoids and predators that are reared on the CY diet should be determined in the fi eld.