5. Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 28 Original Article Interactions between Entomopathogenic Fungus, Metarhizium anisopliae and Sublethal Doses of Spinosad for Control of House Fly, Musca domestica * M Sharififard1, MS Mossadegh2, B Vazirianzadeh3, A Zarei-Mahmoudabadi4 1Department of Medical Entomology, College of Health, Ahvaz Jundishapur University of Medical Sciences, Iran 2Department of Plant Protection, College of Agriculture, Shahid Chamran University, Ahvaz, Iran 3Department of Medical Entomology and Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Science, Iran 4Department of Mycoparasitology and Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Science, Iran (Received 28 Sep 2010; accepted 20 Jul 2011) Abstract Background: Metarhizium anisopliae strain IRAN 437C is one of the most virulent fungal isolates against house fly, Musca domestica. The objective of this study was to determine the interaction of this isolate with sublethal doses of spinosad against housefly. Methods: In adult bioassay, conidia of entomopathogenic fungus were applied as inoculated bait at 105 and 107 spore per gram and spinosad at 0.5, 1 and 1.5 µ g (A.I.) per gram bait. In larval bioassay, conidia were applied as combination of spore with larval bedding at 106 and 108 spore per gram and spinosad at sublethals of 0.002, 0.004 and 0.006 µ g (AI) per gram medium. Results: Adult mortality was 48% and 72% for fungus alone but ranged from 66–87% and 89–95% in combination treatments of 105 and 107 spore/g with sublethal doses of spinosad respectively. The interaction between 105 spore/g with sublethals exhibited synergistic effect, but in combination of 107 spore in spite of higher mortality, the interac- tion was additive. There was significant difference in LT50 among various treatments. LT50 values in all combination treatments were smaller than LT50 values in alone ones. Larval mortality was 36% and 69% for fungus alone but ranged from 58%–78% and 81%–100% in combination treatments of 106 and 108 spore/g medium with sublethals of spinosad respectively. The interaction was synergistic in all combination treatments of larvae. Conclusion: The interaction between M. anispliae and spinosad indicated a synergetic effect that increased the house fly mortality as well as reduced the lethal time. Keywords: Metarhizium anisopliae, Musca domestica, spinosad, Iran Introduction Housefly, Musca domestica L that is well known as poultry and livestock pest is also word-wide mechanical vector of human pa- thogens (Lecouna et al. 2005). High level of insecticide resistance in the housefly and public demands for reducing pesticide use around animal food have promoted interest in the de- velopment of other control strategies of this pest (Geden et al. 1995). An important strat- egy is integrated pest management (IPM) pro- grams, which includes biological, cultural, and/ or chemical methods to control the popula- tion of this pest (Crespo et al. 1998, Lecouna et al. 2005). Although biological control of *Corresponding author: Dr Mona Sharififard, Email: Sharififardm@yahoo.com Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 29 housefly is currently focused mostly on pupal parasitoids, entomopathogenic fungi are ubiq- uitous in nature and could be considered for manipulation in IPM programs (Stainkraus et al. 1990, Barson et al. 1994, 1995, Bywater et al. 1994, Watson et al. 1995, 1996, Renn et al. 1999, Lecouna et al. 2005, Kaufman et al. 2005). The entomopathogenic fungus, Metarhizium anisopliae (Metch) Sorok. has been isolated from 200 insect species including the orders of Lepidoptera, Coleoptera, Orthoptera, and Hemiptera. There are few reports on the use of this fungus for urban pest management (Pachamuthu and Kamble 2000). The major limitations in the use of entomopathogenic fungi such as M. anisopliae have been an extended time to cause sufficient insect mortality and its inconsistent performance under field condi- tions. One of the options for improving the efficacy of the entomopathogenic fungi is to incorporate the fungus pathogens with sub- lethal doses of insecticides (Pachamuthu and Kamble 2000). Data from in vivo compati- bility studies have indicated that M. anisopliae and insecticides are compatible, and their com- bination can have synergistic, antagonistic, or additive effect (Pachamuthu and Kamble 2000, Zurek et al. 2002, Ericsson et al. 2007). Spinosad is a novel macrolide-class insec- ticide produced by the soil bactrium Sac- charopolyspora spinosa and is known to be active against many noxious pests. The me- chanism of action of spinosad appears to be unique, with a primary site of attack being the nicotinic acetycholine receptor and a sec- ondary site of attack possibly being GABA receptors (Scott 1998, Kristensen and Jepersen 2004). In contrast to other commonly used insecticides where the technical active ingre- dients are classified as moderately or high haz- ardous, spinosad is classified as a reduced- risk pesticide and has been determined to pose little to no mammalian toxicity (White et al. 2007). There have been no reports of resistance or cross-resistance in field popula- tion housefly (Scott 1998, Liu and Yue 2000, Kristensen and Jepersen 2004, White et al. 2007). The research objectives were to enhance the lethal effect of M. anisopliae strain IRAN 437C by using it in combination with differ- ent sublethal doses of spinosad against house fly, M. domestica. The aim was to determine which kind of interaction (synergistic, antago- nistic, and additive) occurs between M. ani- sopliae and spinosad and to ascertain the LT50 in control of larvae and adult. Materials and Methods Musca domestica culture Adult house flies were collected from a poultry house by sweeping net and transferred to the laboratory where they were reared at 26° C, 50±5% Rh and photoperiod of 14:10 (L: D). Adults were maintained in cages (40×40× 40 cm3) covered by gauze. Water and food in the form of sugar and powdered milk were provided and replenished every 24–48h. Larval medium comprised 55 g wheat bran, 3g date extract and 2g dried alfalfa suspended in 140 ml water. One cup (250ml volume) of this me- dium was left in each cage for adult oviposi- tion and subsequent development of larvae. The food was replaced every 24–48h. Fungus Ten Iranian isolates of Beauveria bassi- ana (Bals) Vuill. and Metarhizium anisopliae (Metch) Sorok. were obtained as cultures from the Ministry of Jihad Keshavarzy of Iran. Previ- ous study indicated that M. anisopliae strain IRAN 437° C was the most virulent against house fly, M. domestica that caused higher mortality in the shorter time than the others (Sharififard et al. 2011), so this isolate was selected for current study. It was cultured on sabouraud dextrose agar with yeast extract (SDAY) for 2 weeks at 27°C, 75±5% Rh and photoperiod of 12:12 (L:D). Sporulating cul- tures were harvested by scraping the dry co- nidia from the surface of the culture plate Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 30 with a scalpel and transferring them to sterile distilled water containing 0.01% Tween–80. The concentration of the suspension was de- termined using a hemocytometer. Adult Bioassay Spinosad concentrations that caused zero mortality after 48 h in the adult house fly were selected as sublethal based on conduct- ing several pretests. There were 0, 0.5, 1 and 1.5 µ g (AI) per gram bait. Selected M. ani- sopliae concentrations were 0, 105 and 107 conidia per gram bait. Adult bait containing sugar, powdered milk and distilled water were prepared and treated with different combina- tions of spinosad and conidial concentra- tions. Cohorts of twenty-five 2-3 day old house flies were housed in small cage (20×20×20 cm3). Each cage contained a 9 cm diameter Petri dish lined with Watman filter paper and 10 g treated bait. Adults in the control groups were feed with untreated bait. Each treat- ment was replicated 5 times. Cages were main- tained in room conditions and checked daily over a period of 9 days for mortality recording. Larval Bioassay Concentrations of spinosad that produced less than 30% mortality of the larval house- fly larvae were determined using several pre- tests and classified as sublethal doses. There were 0, 0.002, 0.004, and 0.006 µ g (AI) per gram larval bedding. In another treatment, we have also determined that 106 and 108 co- nidia/g larval bedding as sub lethal concen- trations of M. anisopliae strain IRAN 437C in the control of house fly larvae. Plastic 150- ml containers were filled with 50 g larval bedding, containing of wheat bran, dry al- falfa, Date extract and water. The stock sus- pension of fungi was adjusted to a concentra- tion of 0, 5×107 and 5×109 conidia/ml with an improved hemocytometer. One milliliter of each stock fungi suspension was added to each larval container to raise the larval bedding con- centration to 0, 106 and 108 conidia/g bedding. Both spinosad and M. anisopliae treatments were mixed into the larval bedding with a glass rod. In total, the treatments evaluated in this bioassay included 12 different com- binations of the insecticide and fungi concentra- tions. Twenty larvae were used per treatment and each treatment was replicated 4 times. Larva in the control groups were treated with distilled water. However, mortality was ob- served daily for all treatments and the dead larva were removed. Statistical Analysis Data from this study were analyzed by factorial analysis of variance (ANOVA) by us- ing two factor complete randomized design of MSTATC software. Percentage mean of mortality were compared using Duncan's multi- ple range test at α= 0.05. Significant differ- ences among the combination treatments by factorial analysis indicated that there was an interaction between M. anisopliae and insec- ticide and the effect observed might be syn- ergistic or antagonistic. In contrast, if there was no significant difference in M. anisopliae plus insecticide treatment, it implied that the ef- fects were additive (Pachamuthu et al. 2000). Chi-squared tests were performed to deter- mine the type of interaction (additive, syner- gistic or antagonistic). Expected mortality (E) was generated from the following formula: E= Ospin + OMet (1– Ospin), where E is the ex- pected mortality, and OSpin and OMet repre- sent the proportion mortality due to treatments of pure spinosad and pure M. anisopliae, re- spectively. The predicted effects of spinosad and M. anisopliae treatments (E) were com- pared with the observed mortality of the binary treatments (O) with following formula, )({ } E EO 22 −=χ (Ericsson et al. 2007). If the calculated chi-squared value exceeds the tabular value, then it indicates either synergistic or antagonistic interaction. In contrast, if the tabu- lar value exceeds calculated chi- square value, then it indicates an additive effect. LT50 values Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 31 and 95% confidence limits of each value for different treatments were calculated by using probit method of SAS software. When there was no overlap in the 95% CL of lethal time values, the treatments difference were consid- ered significant. Results Adult Bioassay These sub lethal concentrations of spino- sad were classified as 0, 0.5, 1 and 1.5 µ g (AI) per gram bait. The results of analyze vari- ance showed that adult mortality was signifi- cantly affected by insecticide concentration (F= 90.7, df= 3, P< 0.0001), conidial concentration (F= 623.86, df= 2, P< 0.001) and interaction of insecticide and fungi (F=3.19, df= 6, P< 0.011). Higher mortality was observed in M. anisopliae plus spinosad combination treat- ments than sole treatment of fungi or insecti- cide (Table 1). Mixing of 105 conidia/g with 0.5, 1 and 1.5 gµ (AI)/g of spinosad caused higher mortality of adult housefly than alone treatments. Estimation of Chi-squared showed synergistic interaction in combination of 105 conidia/g combined with 1 and 1.5 µg (AI)/g. In the combination treatments of 107 conidia/g with sublethals of spinosad, there was no sig- nificant interaction between insecticide and M. anisopliae. The increased mortality was the result of an additive effect (Table 2). Based on individual treatment levels, the greatest syn- ergistic effect occurred when 105 conidia/g bait were used with 1.5 µ g (AI)/g. Calculated LT50 values and 95% confidence limits of each value for different treatments in adult bioassay showed that the LT50 values were lower in all combination treatments of M. anisopliae+spinosad in comparison with M. anisopliae alone (Table 3). Combination treatments caused faster mortality than the alone ones. When there was no overlap in the 95% CL of lethal time values, the treatments difference were considered significant. While the interaction was additive in the combination of 107 conidia/g with sublethals of spinosad, but there was significant difference in LT50 values between M. anisopliae (107) and M. anisopliae (107) plus spinosad (0.5, 1, 1.5 µg). The shortest lethal time for causing 50% mor- tality in adult population was observed in 107 conidia of M.anisopliae +1.5 µ g of spinosad. There was no significant difference in LT50 val- ues of M. anisopliae (107) + spinosad (0.5 and 1 µ g). There was a significant difference in LT50 be- tween M. anisopliae (105) + spinosad (0.5, 1 and 1.5µ l) and M. anisopliae (105) alone, but there was no difference in the LT50 values among 105 conidia of M. anisopliae +1 and 1.5 gµ of spinosad, also between M. anisopliae (105)+ spinosad (1.5) and M. anisopliae (107)+ spinosad(1.5). Therefore, due to the greatest synergistic effect occurred when 105 conidia were used with 1.5 µ g (AI) of spinosad and no significantly difference in LT50 value of this treatments with M. anisopliae (107) + spino- sad (1.5), mentioned combination of M. ani- sopliae and spinosad was the best combina- tion for control of adult housefly. Larval Bioassay The results of analyze variance showed that larval mortality was significantly affected by insecticide concentration (F= 149.84, df= 3, P< 0.0001), conidial concentration (F= 895.83, df= 2, P< 0.001) and interaction of insecti- cide and fungi (F= 12.78, df= 6, P= 0.025). The percent of mortality of medium size larvae was significantly difference among all 11 treat- ments (Table 4). The greatest mortality was recorded in the combination treatments of 108 spores of M. anisopliae plus sublethals of spinosad. A synergistic interaction between M. anisopliae and spinosad was always found when the fungus was applied at a dosage of 106 and 108 conidia/g larval bedding in com- bination with 0.002, 0.004 and 0.006 µ g (AI)/g of spinosad. But in the combination of 106 spores of M. anisopliae+sublethals of spinosad chi-squared Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 32 values were greater than 108 spores with same sublethal of spinosad (Table 5). The greatest synergetic effect observed when 106 conidia of M. anisopliae were combined with 0.006 µ g (AI), so this was the best combination of M. anisopliae with spinosad for larval control. Table 1. Toxicity of spinosad (µ g (AI)/g) and M. anisopliae (Conidia/g) alone and in combination treatments on adult house fly after 9 days Treatmenta n %Mortality(±SE)b M. anisopliae(105) 150 44±4.20G M. anisopliae (107) 150 72.4±1.79E Spinosad (0.5) 150 21±1.24J Spinosad( 1) 150 32±1.7I Spinosad (1.5) 150 39±1.7H 105+0.5 150 66.4±2.68F 105+1 150 80.6±3.13D 105+1.5 150 87±1.22C 107+0.5 150 89±4.02BC 107+1 150 90.4±1.79B 107+1.5 150 95±3.3A a Each treatment (containing 30 adults) were replicated 5 times. b Means followed by the same letters were not significantly different (Duncan's test;α= 0.05). Table 2. Synergy bioassay: adult house fly mortality from Combination Traetments of Spinosad and M. anisopliae after 9 days Treatment %Mortality Fungi (Conidia/g) Spinosad µg (AI)/g Fungi Spinosad Expected Observed 2χ * 105 0.5 44 21 56 66 1.79 105 1 44 32 62 81 5.82* 105 1.5 44 39 66 87 6.68* 107 0.5 72 21 78 89 1.55 107 1 72 32 81 90 1.00 107 1.5 72 39 83 95 1.73 *A chi-square comparison that exceeds 3.84 with df= 1 and α= 0.05 is considered synergistic and is de- noted by an asterisk (*). Table 3. Calculated LT50 values for M. anisopliae (conidia/ g) and its combination with sublethal doses of spinosad (µ g (AI)/g) bait Treatmenta n Slope±SE LT50 b 95%CLc 2χ (df) M. anisopliae (107) 150 6.9 ± 0.58 6.4 6.12 – 6.67 4.85(2) M. anisopliae 107+Spinosad 1.5 150 4.08 ± 0.33 2.6 2.36 – 2.83 4.91(2) M. anisopliae 107+Spinosad 1 150 3.39 ± 0.31 3.7 3.34 – 4.02 0.96(2) M. anisopliae 107+Spinosad 0.5 150 3.81 ± .032 4.1 3.69 – 4.35 1.05(2) M. anisopliae (105) 150 6.58 ± 1.77 8.08 7.69 – 8.73 8.42(2) M. anisopliae 105+Spinosad 1.5 150 4.37 ± 0.78 3.1 2.71 – 3.55 10.05(2) M. anisopliae 105+Spinosad 1 150 2.71 ± 0.31 3.9 1.84 – 5.96 0.33(2) M. anisopliae 105+Spinosad 0.5 150 2.74 ± 0.27 4.9 4.47 – 5.44 0.94(2) Spinosad 1.5 150 1.74 ± 0.28 12.4 9.59 – 19.6 1.01(2) Spinosad 1 150 2.01 ± 0.32 14.1 10.79 – 22.8 1.03(2) Spinosad 0.5 150 1.74 ± 0.35 21.2 13.98 – 53.17 1.53(2) aEach treatment (containing 30 adults) were replicated 5 times. b Number of days until 50% mortality occured after different treatments. c Treatments will have significant effect on LT50 if there was no overlap of 95% CL. Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 33 Table 4. Toxicity of spinosad (µ g (AI)/g) and M. anisopliae (Conidia/g) alone and in Combination Traetments against house fly larvae Treatmenta n %Mortality(±SE)b M. anisopliae (106) 100 36±1.93 I M. anisopliae 106 +Spinosad 0.002 100 58±2.58 G M. anisopliae 106 +Spinosad 0.004 100 65± 3.42 F M. anisopliae 106 +Spinosad 0.006 100 78±2.5 D M. anisopliae (108) 100 69±1.91 E M. anisopliae 108+Spinosad 0.002 100 81±2.52 C M. anisopliae 108 +Spinosad 0.006 100 95± 1.91 B M. anisopliae 108+Spinosad 0.006 100 100±0.00 A Spinosad 0.002 100 14± 2.58 K Spinosad 0.004 100 23±1.91 J Spinosad 0.006 100 41±1.91 H a Each treatment (containing 25 larvae) were replicated 4 times. b Means followed by the same letters were not significantly different (Duncan's test; α= 0.05). Table 5. Synergy bioassay: larval mortality from combined treatments of Spinosad and M. anisopliae after 9 day Treatment %Mortality Fungi (Conidia/g) Spinosad µg (AI)/g Fungi Spinosad Expected Observed 2χ 106 0.002 35 14 44 58 4.38* 106 0.004 35 23 50 65 9.90* 106 0.006 35 41 62 78 15.75* 108 0.002 69 14 65 81 3.94* 108 0.004 69 23 76 96 5.19* 108 0.006 69 41 82 100 4.09* *A chi-square comparison that exceeds 3.84 with df= 1 and α= 0.05 is considered synergistic and is de- noted by an asterisk (*). Discussion Because conidia require at least 12–24 h for development of germ tube, appressoria and penetration to insect cuticle, so the doses of spinosad that caused <40% mortality 48h after exposure in the adults were selected as sublethals. These doses would allow sufficient time for conidia to form the germ tube and appressoria. High mortality by insecticide dur- ing this period affects the effectiveness of fungus. In our study, the M. anisopliae strain IRAN 437C was effective and caused 44% and 72% mortality in adult population at the concentrations of 105 and 107 spores per gram bait in 9 days after exposure. Synergistic inter- action was observed in combination treat- ments of 105 spore with sublethal doses of insecticide but in combination of 107 spore the interaction was additive. Lethal time in all com- bination treatments were reduced in compari- son with alone treatments of fungi. Thus, in- creased mortality and lowered LT50 values were a general pattern observed in most of Insecticide + M. anisopliae combinations against house fly in our study. In larval test, this fungal strain caused 35 and 69% mortality at 106 and 108 spores per gram bedding in larval population in the end of larval cycle. When spinosad and M. ani- sopliae were applied together as a mixture, larval mortality was significantly higher than the expected value of their additive effect, Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 34 which indicated a synergistic interaction in all treatments. Lower dosages of spinosad not only enhanced the efficacy of M. anisopliae, but also lead to a reduced quantity of inocu- lum needed to cause high levels of mortality in house fly adult and larvae. The time to mortality of larvae could not be accurately assessed as a proportion of infected larvae subsequently died in the pupal stage. More- over, it was not considered in larval bioassay because the eventually aim of larval control is decreasing of adult population and lethal period of larvae is not too important. Earlier studies by Barson et al. (1994), Renn et al. (1999) also demonstrated the ef- fectiveness of M.anisopliae in controlling house fly. In spite of effectiveness of entomopatho- genic fungi against house fly, different strains require different times to achieve high mor- tality. With due attention to high reproduc- tion rate and short life cycle of M. domestica, it is necessary to find approach for increas- ing pest mortality as well as reducing the lethal time by biopesticide agents. So, in this study, we evaluated the effect of combined applications of M. anisopliae and spinosad against M. domestica under laboratory condi- tions. Several studies have focused on the potential use of entomopathogenic fungi in combination with sublethal doses of organic insecticides against various insect pests such as compatibility of M. anisopliae with sub- lethals of chlorpyrifos, propetamphos and cy- fluthrin against the German cockroach (Pacha- muthu et al. 2000), M. anisopliae with Boric Acid against German cockroach (Zurek et al. 2002), combination of Imidiaclopride and Dia- tomaceous Earth with Beauveria bassiana on mole cricket (Thompson et al. 2006), sub- lethals of spinosad with M. anisopliae against exotic wireworms (Ericsson et al. 2007) and M. anisopliae in combination with sublethal doses of imidiacloprid on the subterranean bur- rower bug Cyrtomenus bergi (Jaramillo et al. 2005). Sublethal dosage of synthetic insecti- cides can act as physiological stressors and/ or behavioral modifiers, thereby predispos- ing insects to diseases (Inglis et al. 2001). Integrating insecticides and entomopatho- gens has a few advantages: 1) such approach will increase pest mortality as well as reduce the lethal time, 2) prolong the use of a par- ticular insecticide by reducing the total amount of insecticide using, 3) minimizing environ- mental contamination and increasing human safety, 4) it accelerates the mode of action of fungus without compromising the fungus growth from cadavers that is crucial for inducing epi- zootic in house fly population particularly in larval bedding that humidity and temperature of bed supported the growth of muscardine on larval cadavers. In conclusion, our results indicated that the use of combination of M. anisopliae with lower dosage of spinosad might become an important component of M. domestica IPM but at first, this approach must be testing un- der field conditions. Acknowledgements The authors wish to thanks Dr Rassule Zare from Iranian Research Institute of Plant Protection for providing the fungal isolates used in the study and for his useful advice. The authors also thank Research Deputy of Shahid Chamran University, Ahvaz, Iran for sponsoring this project. The authors declare that there is no conflict of interests. References Barson G, Renn N, Bywater AF (1994) Laboratory evaluation of six species of entomopathogenic fungi for control of House fly Musca domestica L, a pest of intensive animal units. J Inverter Pathol. 64: 107–113. Bywater AF, Barson G, Renn N (1994) The potential of oil-based suspension of Metarhizium anisopliae conidia for the control of the housefly (Musca domes- Iran J Arthropod-Borne Dis, 2011, 5(1): 28–36 M Sharififard et al.: Interactions between … 35 tica), a pest of intensive animal units. In: Proc. Brighton Crop Protection Conf. The British Crop Protection Council, Farnham, UK, 3: 1097–1102. 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