1. Introduction
Egg parasitoids effectively manage a number of crop pests (Moura et al., 2006), and thus regulate natural populations of other insects (Delpuech and Delahaye, 2013). The parasitic wasps belonging to the genus Trichogramma are widely distributed and perform key role in the control of the pests belonging to the order Lepidoptera on a wide range of agro-ecosystem (Hassan and Abdelgader, 2001). Pesticides use adversely affects the beneficial performance of Trichogramma (King et al., 1986; Khan et al., 2015b). Therefore, it is required to evaluate pesticides adverse impacts on Trichogramma to effectively integrate both chemical and biological controls (Khan et al., 2015a).

Trichogramma chilonis (Ishii) have wide distribution throughout the Indian subcontinent and has been used to effectively control caterpillar pests in various agro-ecosystems (Manjunath et al., 1985; Khan et al., 2014). They control pests including Chilo spp. in sugarcane, maize, and Helicoverpa armigera in cotton, tomato, and lady’s finger in India (Singh, 2001). It is an important egg parasitoid of lepidopteran pests in Pakistan (Sattar et al., 2011; Khan et al., 2014), and control pests including sugarcane borer (Chilo sacchariphagus) in sugar cane, diamondback moth (Plutella xylostella) in cabbage and other vegetables, and cotton bollworms (Helicoverpa armigera) in cotton and corn (Rasool et al., 2002), and rice leaffolder Canphalocrocis medinalis (Guenée) in Pakistan (Sagheer et al., 2008).

The current study aimed to evaluate the adverse effects of lower doses of fipronil (≤ 0.4x) in the field on the larval and pupal mortalities of T. chilonis within the host eggs, and effects on parasitism of previously treated eggs of Sitotroga cerealella by the minute parasitoid in order to determine the rate of the chemical most compatible with T. chilonis.

2. Materials and Methods
2.1. Rearing of Sitotroga cerealella Olivier on Wheat Grain
Eggs of grain moth S. cerealella were sprinkled on the wheat grain and the young larvae hatched within 4-6 days, and were shifted to plastic rearing jars (15x 20 cm), maintained in the laboratory at average conditions of 24 ± 6οC, 65 ± 10% relative humidity (RH) and 16:8 (L:D) for 20-25 days until adults emerged.

Regular collections of emerged moths into oviposition jar (10 x15 cm in dimension) were made. The jar was placed over the corn flour in a metal/plastic tray, for purpose of egg laying for 24 h. Subsequently, the host eggs were collected by sieving the flour, and were used for the rearing and in experiments.
 
2.2. Rearing of Trichogramma chilonis (Ishii) on S. cerealella
Approximately 1000-1300 fresh host eggs were glued to paper card (5 x 8 cm), and card was allowed to dry for 1-2 hrs. Subsequently, the dried card was exposed to parasitism for 24 h. in glass jars (5x12 cm) containing approximately 30 to 40 adults (mixed- gender) of T. chilonis, under the lamp light. The likely parasitized card was removed and was transferred to another glass jar, and was incubated at the 23±3οC, 70±10% RH and 14:10 (L:D) conditions until adult emerged. The stock culture of T. chilonis was maintained and was used in the subsequent experiments

2.3. Preparation of Different Concentrations of Pesticides Solution
Commercially available fipronil was diluted with tap water to prepare stock solution of field recommended dose (x = 480 ml/acre) under the aforementioned laboratory conditions. The stock solution was diluted (serial dilutions) and 5 different concentrations including 0.4x, 0.2x, 0.08x, 0.04x, and 0.02x of insecticide were prepared for use in the experiments by the formula: C1V1 = C2V2, where C1 and V1 are the concentration and volume of commercial pesticides/stock solution, respectively, while C2 and V2 are the concentration and volume of the required pesticide solutions (diluted), respectively.

2.4. Testing of Pesticides against Larval and Pupal Stages of T. chilonis
Approximately 200-300 fresh S. cerealella eggs card was exposed for 24 hours to parasitoids in glass jars (5x12 cm) containing approximately 14 to 20 mixed -gender adults of T. chilonis under aforementioned laboratory conditions. The likely parasitized card was subsequently removed and was incubated under conditioned mentioned earlier until larvae (three days old parasitized) and pupae (six days old parasitized) were formed, followed by the card was removed, and was cut to small card strips (approximately 0.8 x 8 cm), each containing 10-15 host eggs, prepared separately for each aforementioned doses. For larval testing, three days (72 h), while for pupal stage treatment six days (144 h) old parasitized cards were dipped for 1-2 seconds in the pesticide solution or in water (untreated: control) of each treatments (stage). The trial consisted of dipping ten strip cards for each stage, in order to assess dose-wise and stage-wise effects of pesticides on T. chilonis regarding emergence. Subsequently card was removed and air dried at room temperature for 1 h. Each dried, parasit- ized card strip was transferred into a vial (1x10 cm), and was incubated at aforementioned controlled cond- itions until adult emerged.

The number of pupae of the pesticides dipped card, and the number of the specimens emerged from the pupae were determined by magnifying lens without dissecting them (generally one T. chilonis adult emerged from single parasitized S. cerealella eggs), and recorded separately for each concentration and stage treated with pesticide.

2.5. Testing the Pesticides to Assess Parasitism by T. chilonis
Approximately 100 to 175 fresh S. cerealella eggs were glued on hard paper card (5x8 cm), and was dried, subsequently cut into 10 strips (0.9x8cm each) each of approximately 20-35 host eggs, and were followed dipped for 1-2 seconds in the solution of each of aforementioned doses in addition to water. The cards were dried, and were transferred to a glass vial (1x10 cm) containing one pair of T. chilonis (< 24 h old) for 24 h for completion of parasitisation of previously treated host eggs. The females were removed and the vials were incubated at aforementioned conditions until pupae formation. The data were recorded by counting pupae after 7 days of exposure to parasitoids.

2.6. Data Analysis
The recorded data were analyzed using general linear model (Statistix 9) on percent emergence as well as average parasitization. Mean separation was carried out by Tukey HSD test (P = 0.05). Reduction in emergence (%) or reduction in parasitism (%) over controls were evaluated by toxicity categories of International Organization for Biological Control (IOBC)/West Palaearctic Regional Section (WPRS) (Hassan et al., 1994; Sterk et al., 1999): 1= harmless (E < 30%); 2 = slight harmful (30 ≤ E ≤ 79%); 3 = moderately harmful (79 < E ≤ 99%); 4 = harmful ( > 99 %), where “E” stand for effect of the pesticide on the biological control agent measured as the reduction in percentage of emergence or parasitism over control.
 
3. Results
Analysis of variance for dose demonstrated significance difference regarding emergence and parasitism (Table 1 and 2). Emergence from the immature stages demonstrated increase in rate of emergence through decrease in concentration of fipronil (Table 1). All used doses in the larval treatment were significantly different (p≤ 0.05) from their control treatments, while in the pupal treatment, both 0.04x and 0.02x doses showed statistically at par (p> 0.05) with respective control treatments.

In the Table 1 and Figure 1, fipronil demonstrated reduced toxicity for emergence of parasitoids when dose shifted from higher concentration toward lower concentration. fipronil in the current studies was revealed as harmful at 0.4 x as well as 0.2 x doses, moderately harmful at both 0.08x and 0.04x doses, while slightly harmful for emergence of T. chilonis when larval stage of parasitoids was treated at 0.02x of the field dose. However, when pupae of parasitoids were treated, 0.4x dose showed as moderately harmful, both 0.2x and 0.08x doses were slightly harmful, while, the remaining two doses demonstrated as harmless for the emergence of parasitoids. T. chilonis The results in Table 2 showing increase in mean parasitism rate when dose shifted from 0.2x toward 0.02x, and also indicated that all the used doses were slightly harmful for parasitism except 0.02x dose, which was harmless for parasitism. Similarly, percentage parasitism relative to control (Figure 2) of the previously treated eggs of S. Cerealella revealed that 0.02x dose showed good mean parasitism of 87.7% relative to control, while the remaining doses showed mean parasitism approximately ≤ 70% relative to control.

Figure 1 Percentage reduction in emergence over control of T. chilonisfrom the host eggs of S. cerealellatreated with fipronil at larval and pupal stages of parasitoids

Figure 2 Percentage parasitism relative to control of host eggs of S. cerealella by T. chilonis previously treated with fipronil

Table 1 Percentage emergence (mean ± SE) of T. chilonis from eggs of Sitotroga cerealella Olivier treated with fipronil when parasitoids were at larval and pupal stages, and means comparison based on Tukey HSD, P = 0.05 or 5%

Table 2 Parasitism (mean ± SE) of previously treated eggs of Sitotroga cerealella Olivier with fipronil by T. chilonis, and means comparison based on Tukey HSD, P = 0.05 or 5%

 
4. Discussion
In this present studies, fipronil was proved to be one of the most harmful chemical for emergence as well as parasitism by the females emerged from the treated host eggs used in the agro-ecosystem. This chemical showed severe lethal as well as sublethal effects on T. chilonis even at lower doses of half field dose (< 0.5x) including 0.4x and 0.2x doses. For successful integration with Trichogramma or any other natural Figure 2 Percentage parasitism relative to control of host eggs of S. cerealella by T. chilonis previously treated with fipronil enemies in particular agro-ecosystem requires more studies under field condition. Furthermore, it should be noted that the repeated use of this lower dose might be responsible for the development of resistance in pest species.

Sufficient literature is not available on fipronil toxicity to Trichogramma or any other parasitoids. However, there were few studies to assess the adverse impacts of the chemical at field dose on survival of adults Trichogramma and their parasitism of host eggs in the previous literature. Khan et al. (2015b) found that fipronil was slightly harmful at field dose for parasitism of the previously treated host eggs of S. cerealella by T. chilonis. Moreover, Khan et al. (2015a) investigated that fipronil induces approximately 100% mortality of adult T. pretiosum at field dose within 24 hours of exposure to treated cotton leaves.

Fipronil is a broad-spectrum insecticide that belongsto the family phenylpyrazole, and has been in commercial use since 1993 (Tingle et al., 2003). It prevent the passage of chloride ions through the GABA (gamma aminobutyric acid) receptor and glutamate-gated chloride (GluCl) channels, components of the central nervous system Colliot et al., 1992; Cole et al.,1993; Tingle et al., 2003), and in such way interferes with the insect central nervous system, which led to hyperexcitation of nerves and muscles of insects. It is commonly used in rice and sugarcane to control stem borers at field recommended concentration of 480 ml/acre, and is available with trade name “Regent” 5% SC in Pakistan.

The recommended rate of fipronil (4%) was very effective against Hieroglyphus spp., in rice, maize, wheat, and sugarcane in Pakistan (Sultana and Wagan, 2011). The early stage nymphs of various stages of Hieroglyphus perpolita (Uvarov), H. oryzivorus Carl and H. nigrorepletus I. Bolivar under laboratory conditions were more susceptible for fipronil compared to later stage nymphs or the adults (Sultana and Wagan, 2011). Fipronil applied at the rate of 0.6 g a.i./ha in the field successfully suppressed outbreaks of adult grasshoppers, including Oedaleus senegalensis Krauss and Acrotylus blondeli Saussure, with 47%  mortality obtained in 2 days and 91% in 10 days (Balanca and Visscher, 1997).  Low doses of fipronil (5-20 g a.i. ha-1) effectively controlled the brown locust, Locustuna purdalina (Walker), the African migratory locust, Locusta migratoria migratorioides (Reiche & Fairmaire), and the desert locust in laboratory and field tests, (Butler and Du Preez, 1994; Kriel et al., 1994; Megenasa and Muinamia, 1994; Price et al., 1994). Fipronil also contributed to the management of populations of the Colorado potato beetle Leptinotarsa decemlineata (Say) in another study (Shi et al., 2012).
 
Conclusion
Fipronil is one of the most toxic chemicals for Trichogramma used in the agro-ecosystem. It exhibited high toxicity for emergence of adults as well as parasitism by Trichogramm chilonis even at lower doses such as both 0.4x and 0.2x doses.
 
Acknowledgments
The 5000 indigenous PhD fellowship provided by the Higher Education Commission of Pakistan to the first author is strongly acknowledged. The authors are also grateful to the administration of Nuclear Institute of Food and Agriculture (NIFA), Tarnab Farm, Peshawar for the provision of the laboratory facilities for conduct of research. Dr. Hizbullah Khan of University of Peshawar is also acknowledged for their help in obtaining permission to work in the NIFA.
 
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