Interactions between Entomopathogenic Fungus, Metarhizium anisopliae and Sublethal Doses of Spinosad for Control of House Fly, Musca domestica

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 μg (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 μg (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 interaction 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.

Introduction

Housefly, Musca domestica L that is well known as poultry and livestock pest is also word-wide mechanical vector of human pathogens (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 development of other control strategies of this pest (Geden et al. 1995). An important strategy is integrated pest management (IPM) programs, which includes biological, cultural, and/or chemical methods to control the population of this pest (Crespo et al. 1998, Lecouna et al. 2005). Although biological control of housefly is currently focused mostly on pupal parasitoids, entomopathogenic fungi are ubiquitous in nature and could be considered for manipulation in IPM programs (Stainkraus et al. 1990, Barson et al. 1994, Barson et al. 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 conditions. One of the options for improving the efficacy of the entomopathogenic fungi is to incorporate the fungus pathogens with sublethal doses of insecticides (Pachamuthu and Kamble 2000). Data from in vivo compatibility studies have indicated that M. anisopliae and insecticides are compatible, and their combination 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 insecticide produced by the soil bactrium Saccharopolyspora spinosa and is known to be active against many noxious pests. The mechanism of action of spinosad appears to be unique, with a primary site of attack being the nicotinic acetycholine receptor and a secondary site of attack possibly being GABA receptors (Scott 1998, Kristensen and Jepersen 2004). In contrast to other commonly used insecticides where the technical active ingredients are classified as moderately or high hazardous, 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 population 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 different sublethal doses of spinosad against house fly, M. domestica. The aim was to determine which kind of interaction (synergistic, antagonistic, and additive) occurs between M. anisopliae 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 medium was left in each cage for adult oviposition and subsequent development of larvae. The food was replaced every 24–48h.

Fungus

Ten Iranian isolates of Beauveria bassiana (Bals) Vuill. and Metarhizium anisopliae (Metch) Sorok. were obtained as cultures from the Ministry of Jihad Keshavarzy of Iran. Previous 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 cultures were harvested by scraping the dry conidia from the surface of the culture plate with a scalpel and transferring them to sterile distilled water containing 0.01% Tween–80. The concentration of the suspension was determined 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 conducting several pretests. There were 0, 0.5, 1 and 1.5 μg (AI) per gram bait. Selected M. anisopliae 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 combinations of spinosad and conidial concentrations. 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 treatment was replicated 5 times. Cages were maintained 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 housefly larvae were determined using several pretests and classified as sublethal doses. There were 0, 0.002, 0.004, and 0.006 μg (AI) per gram larval bedding. In another treatment, we have also determined that 10⁶ and 10⁸ conidia/g larval bedding as sub lethal concentrations 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 alfalfa, Date extract and water. The stock suspension of fungi was adjusted to a concentration 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 concentration 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 combinations of the insecticide and fungi concentrations. 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 observed 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 using two factor complete randomized design of MSTATC software. Percentage mean of mortality were compared using Duncan's multiple range test at α= 0.05. Significant differences among the combination treatments by factorial analysis indicated that there was an interaction between M. anisopliae and insecticide and the effect observed might be synergistic or antagonistic. In contrast, if there was no significant difference in M. anisopliae plus insecticide treatment, it implied that the effects were additive (Pachamuthu et al. 2000). Chi-squared tests were performed to determine the type of interaction (additive, synergistic or antagonistic). Expected mortality (E) was generated from the following formula: E= O_spin + O_Met (1– O_spin), where E is the expected mortality, and O_Spin and O_Met represent the proportion mortality due to treatments of pure spinosad and pure M. anisopliae, respectively. The predicted effects of spinosad and M. anisopliae treatments (E) were compared with the observed mortality of the binary treatments (O) with following formula, $\raisebox{1ex}{${\chi }^2=\{{(O-E)}^2\}$}\!\left/ \!\raisebox{-1ex}{$E$}\right.$ (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 tabular value exceeds calculated chi-square value, then it indicates an additive effect. LT₅₀ values 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 considered significant.

Results

Adult Bioassay

These sub lethal concentrations of spinosad were classified as 0, 0.5, 1 and 1.5 μg (AI) per gram bait. The results of analyze variance showed that adult mortality was significantly 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 treatments than sole treatment of fungi or insecticide (Table 1). Mixing of 10⁵ conidia/g with 0.5, 1 and 1.5 μg (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 significant 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 synergistic effect occurred when 10⁵ conidia/g bait were used with 1.5 μg (AI)/g.

Table 1.

Toxicity of spinosad (μg (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

^aEach treatment (containing 30 adults) were replicated 5 times.

^bMeans 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	χ2*
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 denoted by an asterisk (*).

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% mortality in adult population was observed in 10⁷ conidia of M.anisopliae +1.5 μg of spinosad. There was no significant difference in LT₅₀ values of M. anisopliae (10⁷) + spinosad (0.5 and 1 μg).

Table 3.

Calculated LT₅₀ values for M. anisopliae (conidia/g) and its combination with sublethal doses of spinosad (μg (AI)/g) bait

Treatmenta	n	Slope±SE	LT50b	95%CLc	χ2 (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.

^bNumber of days until 50% mortality occured after different treatments.

^cTreatments will have significant effect on LT₅₀ if there was no overlap of 95% CL.

There was a significant difference in LT₅₀ between M. anisopliae (10⁵) + spinosad (0.5, 1 and 1.5μl) and M. anisopliae (10⁵) alone, but there was no difference in the LT₅₀ values among 10⁵ conidia of M. anisopliae +1 and 1.5 μg 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 μg (AI) of spinosad and no significantly difference in LT₅₀ value of this treatments with M. anisopliae (10⁷) + spinosad (1.5), mentioned combination of M. anisopliae and spinosad was the best combination 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 insecticide and fungi (F= 12.78, df= 6, P= 0.025). The percent of mortality of medium size larvae was significantly difference among all 11 treatments (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 combination with 0.002, 0.004 and 0.006 μg (AI)/g of spinosad.

Table 4.

Toxicity of spinosad (μg (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

^aEach treatment (containing 25 larvae) were replicated 4 times.

^bMeans followed by the same letters were not significantly different (Duncan's test; α= 0.05).

But in the combination of 10⁶ spores of M. anisopliae+sublethals of spinosad chi-squared 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 μg (AI), so this was the best combination of M. anisopliae with spinosad for larval control.

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	χ2
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 denoted 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 during 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 interaction was observed in combination treatments of 10⁵ spore with sublethal doses of insecticide but in combination of 10⁷ spore the interaction was additive. Lethal time in all combination treatments were reduced in comparison with alone treatments of fungi. Thus, increased 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. anisopliae were applied together as a mixture, larval mortality was significantly higher than the expected value of their additive effect, 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 inoculum 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. Moreover, 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 effectiveness of M.anisopliae in controlling house fly. In spite of effectiveness of entomopathogenic fungi against house fly, different strains require different times to achieve high mortality. With due attention to high reproduction rate and short life cycle of M. domestica, it is necessary to find approach for increasing 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 conditions. 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 sublethals of chlorpyrifos, propetamphos and cyfluthrin against the German cockroach (Pachamuthu et al. 2000), M. anisopliae with Boric Acid against German cockroach (Zurek et al. 2002), combination of Imidiaclopride and Diatomaceous Earth with Beauveria bassiana on mole cricket (Thompson et al. 2006), sublethals 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 burrower bug Cyrtomenus bergi (Jaramillo et al. 2005). Sublethal dosage of synthetic insecticides can act as physiological stressors and/or behavioral modifiers, thereby predisposing insects to diseases (Inglis et al. 2001).

Integrating insecticides and entomopathogens has a few advantages: 1) such approach will increase pest mortality as well as reduce the lethal time, 2) prolong the use of a particular insecticide by reducing the total amount of insecticide using, 3) minimizing environmental 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 epizootic 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 under field conditions.