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Combined Effects of Beauveria bassiana (Hypocreales: Clavicipitaceae) and Insecticide Mixtures on Biological Parameters of Musca domestica (Diptera: Muscidae).

Byline: Muzammil Farooq and Shoaib Freed

Abstract

The current study was conducted to evaluate the effect of Beauveria bassiana (Balsamo) Vuillemin in mixtures with six different insecticides separately against Musca domestica (L). Preliminary experiments were conducted to determine the mortality rates caused by the application of fungal inoculums and different doses of insecticides separately on laboratory reared M. domestica populations. Later, these fungal inoculums and insecticides in mixtures were applied on adults of M. domestica using the bait method. Flies showed concentration/dose dependent response and insecticides i.e. acetamiprid, emamectin benzoate, imidacloprid and lufenuron showed higher mortality in combination with insect pathogenic fungi than expected with significantly synergistic interactions.

The effects of insect pathogenic fungi and insecticides mixtures were assessed on the biological parameters i.e., longevity, fecundity, egg hatching, larval duration, percent pupation, pupal weight, pupal duration, adult emergence and sex ratio of surviving M. domestica populations. The results showed significant effects of pathogenic fungi and insecticides mixtures on all parameters (P<0.05). As a result of application of fungi and insecticide mixtures, a significant decrease in longevity, fecundity, egg hatching, percent pupation, pupal weight and adult emergence was observed, while larval duration and pupal period were prolonged. The entomopathogenic fungi integrated along with insecticides i.e., acetamiprid, emamectin benzoate, imidacloprid and lufenuron could be a viable option for establishing an integrated pest management program for managing populations of M. domestica.

Key words: Beauveria bassiana, Musca domestica Biological aspects Sublethal effects

INTRODUCTION

The house fly, Musca domestica (L.) (Diptera: Muscidae), is a well-known pest and serious threat to human and livestock health by acting as a mechanical vector of pathogens that causes diseases in man and animals (Khan and Ahmed, 2000; Lecouna et al., 2005). In addition to disease transmission, M. domestica causes food spoilage and adults can be a source of nuisance (Ande, 2001; Forester et al., 2009). As human's and livestock health both are put at risk by this pest (Saleh and Elmosa, 2002), therefore, it is critical to control this pest, which is chiefly done by the use of conventional insecticides (Cao et al., 2006; Malik et al., 2007). However, due to the indiscriminate use of insecticides, serious problems like insecticide resistance and residual effects of chemicals are on the rise. Biological control could be promising and eco-friendly alternative for the control of M. domestica (Mishra et al., 2011).

In comparison to insecticides, entomopathogenic fungi provide the potential for the management of M. domestica due to its natural prevalence of in M. domestica populations (Skovgard and Steenberg, 2002; Khan et al., 2012).

Entomopathogenic fungi, such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikof) Sorokin result in rapid killing and high infection rates to control M. domestica (Watson et al., 1995; Lecouna et al., 2005; Kaufman et al., 2005; Sharififard et al., 2011a). In these studies ultimate mortality of M. domestica populations was reached within a time period of 5-15 days. To improve the efficacy of biological control, fungal pathogens can be incorporated with lower doses of insecticides (Pachamuthu and Kamble, 2000). Several studies have indicated that combined usage of insecticides and entomopathogenic fungi such as M. anisopliae are compatible approaches (Pachamuthu and Kamble, 2000; Zurek et al., 2002; Ericsson et al., 2007).

It is not clear how the combination of insecticides and entomopathogenic fungi treatments interact but the physiological effects caused by one agent may increase the effects of the other resulting in higher mortality. The above mentioned studies also indicate that such combined formulations of entomopathogenic fungi and insecticides make less use of active ingredient than what applied separately to achieve the same outcome. These combined approaches interact synergistically and result in significantly higher mortality (Ericsson et al., 2007). The purpose of this study was to determine the effect of different concentrations of B. bassiana and different doses of insecticides against the M. domestica.

Additionally, the effects of combined mixtures of B. bassiana concentrations and insecticides doses on different biological parameters i.e., longevity, fecundity, egg hatching, larval duration, percent pupation, pupal weight, pupal duration, adult emergence and sex ratio among surviving members of M. domestica population was also studied.

MATERIALS AND METHODS

Musca domestica culture

Adult flies were collected from a poultry house and transferred to the Laboratory of Insect Microbiology and Biotechnology, Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan, where flies were reared at 26+-2degC, 50+-5% RH (relative humidity) and photo period (12:12) h in cages (30x30x30cm3) with mesh screen on the opposite sides and a cloth sleeve opening in the front. Adults were provided food in the form of sugar and powdered milk (3:1) in a Petri dish (9 cm) and water, while the larval medium (egg laying medium) was a water based mixture of wheat bran, rice meal, yeast, sugar and dry milk powder (40:10:3:3:1) as reported by Bell et al. (2010) with slight modifications. The diet was changed after 2-4 days depending on the number of larvae present. In order to establish a laboratory population, the flies were reared up to fourth generation then later 2-3 days old adults were used for bioassays.

Entomopathogenic fungi

Two different isolates of B. bassiana (Table I) that caused maximum mortalities in a preliminary experimentation were selected (Data not shown). The slants of monoconidial cultures of the isolates were cultured on potato dextrose agar (PDA) at 25degC in darkness and then stored at 4degC until needed. For further propagation, the spores from these slants were spread on to the surface of PDA plates (9 cm diameter) and kept at 25degC in darkness at 70-75% RH for 14 days (Freed et al., 2011a, b). After 14 days of growth, the fungal spores were used to treat M. domestica or stored at 4degC until used for this bioassay. The conidia were scraped from the plates and mixed with a sterile Tween-80 (0.05%) solution, while the conidial concentration was determined by haemocytometer for further feeding experiments. Conidial viability was determined by enumerating the percentage of germinated conidia 24 h after spreading in fresh PDA medium.

A conidial suspension of 1x107 spores/ml (0.01 ml) was spread on 9cm perti plate containing 15 mL of PDA medium and incubated at 27 degC for 24 h for germination. Three 15mm square cover slips were placed on the surface of medium, and percentage germination was determined by counting the number of germinated conidia and total number of conidia per field view at 250X magnification (Quesada- Moraga et al., 2006). The conidial germination was above 95% (Data not shown).

Entomopathogenic fungi bioassay

Different concentrations of conidial suspensions (3x108, 2x108, 1x108, 1x107 and 1x106 spores ml-1) of B. bassiana isolates (Bb-01 and Bb-08) were prepared by serial dilution with 0.05% Tween 80. Conidial suspension (1 ml) was dispersed on surface of adult diet (1g) (sugar and powdered milk, 3:1) for better distribution of spores. In control, flies were provided with diet containing Tween 80 (0.05%) solution only and water was provided as ad libitum to all flies. Forty adults (2-3 days old) with sex ratio (1:1) were treated separately with each concentration which was replicated four times and placed in jars (15x6x6 cm3) at 26+-2degC, 50+-5% RH. The mortality data was recorded every 24 h for seven consecutive days. Different levels of lethal concentrations (LC10, LC30 and LC50) necessary to kill different percentages of fly population (i.e., 10%, 30% and 50%) were experimentally determined for each fungal isolate (Table I).

Insecticide bioassay

Six different formulated insecticides (Table 1) were used for comparison with insect pathogenic fungi. The insecticides were chosen randomly as these were recommended to control M. domestica population in the field. Preliminary experimentation was done for the measurement of LC10, LC30 and LC50 against M. domestica population for each insecticide separately. The procedure used for the measurement of lethal concentration of insecticides was the same as for the application of fungi.

Effects of fungi and insecticides on biological parameters of the adult M. domestica 500ul of each fungus and insecticide (collectively 1 ml) was added to the 1g of diet (in ratio of 1:1) for adult's treatment, while in the control group insects were provided with diet containing 0.05% Tween 80 and water. 500ul of each fungi and insecticide was adjusted to get the final desired concentration in the diet.

Table I. - Different isolates of Beauveria bassiana and insecticides with LC10, LC30 and LC50 values used against laboratory reared house flies.

Fungal Species###Source###LC10 (spores ml-1)###LC30 (spores ml-1)###LC50 (spores ml-1)

B. bassiana (Isolate Bb-01)###Cotton field###5.71x105###9.32x105###3.78x106

###(Multan, Punjab)

B. bassiana (Isolate Bb-08)###Pine forest soil###1.03x106###5.53x106###9.05x106

###(Mansehra, KPK)

Insecticides###Manufacturer###LC10 (ppm)###LC30 (ppm)###LC50 (ppm)

Acetamprid (20SP)###Arysta Life Sciences###0.03###0.14###0.39

Bifenthrin (10EC)###FMC United###0.02###0.08###0.22

Emamectin benzoate (019) EC###Syngenta###0.00002###0.0002###0.001

Fipronil (5EC)###Bayer Crop Sciences###0.00003###0.0004###0.002

Imidacloprid (20SL)###Bayer Crop Sciences###0.022###0.09###0.27

Lufenuron (050EC)###Syngenta###0.00002###0.0002###0.001

The LC10, LC30 and LC50 of fungus were taken as three different levels of concentrations. On the other hand, LC10, LC30 and LC50 of insecticides were considered as different dose levels for combination with entomopathogenic fungi. LC10 of fungus and LC10 of one insecticide were taken as one treatment, LC30 of fungus and LC30 of insecticide as second and LC50 of fungus and LC50 of insecticide as third for each insecticide in combination with fungal isolate (Bb-01). The same experimental scheme was applied to isolate Bb-08 and all other insecticides. The experiment was carried out on forty adults (sex ratio 1:1) with 4 replications in each treatment and placed in jars (15x6x6 cm3). The mortality data was recorded for seven consecutive days. Insects were provided with same rearing conditions as described earlier.

In addition to mortality, the sublethal effects of different fungi and insecticides mixtures on surviving M. domestica were observed. The male and female adults were provided with oviposition medium, longevity of each sex was determined according to the method by Fletcher et al. (1990). The oviposition medium (described earlier) was examined daily for egg laying and subsequent counting with aid of hand lens. The medium was changed after every two days depending upon the number of eggs. The total number of eggs was recorded and fecundity was calculated, while percent fecundity was determined according to Crystal (1964) by dividing total number of eggs laid to total number of female over the entire experiment. After counting, the eggs were again left in egg laying medium for hatching, which was checked on daily basis for hatching if any. The larvae on hatching were counted and percent hatching was calculated.

The larvae were provided with food and kept until pupation to estimate larval duration. The resultant pupae were separated from larval medium and counted for percent pupation and pupal weight was also recorded. Later pupae were kept separate in jars until the emergence of adults. The adult emergence was measured in accordance to Khazanie (1979), while number of males and females were recorded in order to calculate sex ratio.

Data analysis

The control mortality ranged from 5% to 10%, the observed mortality data was corrected by using Abbott's formula (Abbott, 1925). Later, a statistical program POLO-PC (Lerora Software, 2003) was used to measure different lethal concentrations for each fungal isolate and lethal doses for insecticides separately. Similarly, mortality data for fungi and mixtures was also corrected as explained above. The synergetic effect to mortality of M. domestica by combine use of fungi and insecticides mixtures was analyzed by comparing mortality rates induced by fungi and insecticide mixture (observed) with sum of mortalities caused by fungi and insecticides individually (expected). Following formulae was used for measurement of expected mortality

Me = Mf + Mi (1 - Mf/100),

where Mf and Mi were the observed percent mortalities caused by the fungus and the insecticide separately (Farenhorst et al., 2010). Paired samples T-test in Statistix 8.1 was used for pair-wise comparisons between each treatment and to eliminate potential treatment variations i.e., differences between fungi applications and insecticides effectiveness. Positive Mfi-Me values were considered synergistic (Koppenhofer and Kaya, 1998).

The entire experiment was performed twice for confirmation of results and to avoid ambiguity. The means for longevity, fecundity and other parameters were analyzed with the help of analysis of variance (ANOVA) and separated with the help of LSD at significance level of 5% using Statistix 8.1 software.

RESULTS

Mortality after individual and binary application of fungi and insecticides

Mortality of M. domestica population after application of different fungal isolates, insecticides, individually and mixtures of entomopathogenic fungi and insecticides at different concentrations levels are presented in (Table I, II). The toxicity of tested insecticides in combination with fungi showed impacts on the survival of M. domestica population. The mortality rate was higher in insecticides and fungus mixtures as compared to those caused by insecticides and fungi alone while an increasing trend was found towards higher concentration in a dose dependent manner. In addition, insecticides i.e., acetamiprid, emamectin, imidacloprid and lufenuron showed higher mortality than expected when combined with fungi and effects seemed to be synergetic for isolates (Bb-01, Bb-08) of insect pathogenic fungi due to positive Mfi-Me values after seven days of treatment (Table I).

In the case of a mixture of insect pathogenic fungi (Bb-01) and insecticides, the highest percent mortality (+-SE) 91.43 (+- 0.64) was observed in case of combined use of higher dose of isolate Bb-01 (LC50, 3.78x106 spores ml-1) and acetamiprid (LC50, 0.3 ppm) followed by LC50 of isolate Bb-01 and LC50 of emamectin, (87.28 +- 2.64). These findings were significantly higher as compared to other treatments. The results regarding entomopathogenic fungi (LC30) and insecticides (LC30) mixture, showed highest percent mortality (63.80 +- 0.90) in isolate Bb-01(LC30) and emamectin (LC30). Moreover, in case of LC10 of insect pathogenic fungi and LC10 of insecticide, highest percent mortality (30.84 +- 0.52) was observed for isolate Bb-01(LC10) and acetamiprid (LC10).

Similar result were also observed for insect pathogenic fungi (Bb-08) and insecticides mixture where highest percent mortality (+-SE) 87.02 (+-1.08) was observed in case of combined use of higher dose of isolate Bb-08 (LC50) and acetamiprid (LC50). Moreover, (LC30) of Bb-08 and insecticides (LC30) mixture, showed highest percent mortality (43.26 +- 1.35) when isolate Bb-08(LC30) was combined with acetamiprid (LC30). While, in case of LC10 (Bb-08) and LC10 of insecticide, highest percent mortality (28.14 +- 0.67) was observed for isolate Bb-08(LC10) and acetamiprid (LC10).

Combined effects of fungi and insecticides Longevity

In general, results indicated that male longevity decreased significantly as a result of treatment with fungi and insecticides mixtures especially at higher concentrations (Figs. 1a, b). Male longevity significantly varied among all treatments. A significant reduction was observed in male longevity at LC50 of isolate Bb-01 and LC50 of emamectin, reducing male longevity (+-SE) of M. domestica to 7.83 (+- 0.44) days followed by 8.18 (+-0.41) days in LC50 of isolate Bb-01 and LC50 of lufenuron far <all treatments as compared to the control (F=13.25, P <0.0001) (Fig. 1a).

Similar results were observed in case of Bb-08 where maximum reduction in male longevity was observed at LC50 of isolate Bb-01 and LC50 of emamectin reducing male longevity to 9.13 (+-0.54) days (F=6.43, P <0.0001) (Fig. 1b).

In female longevity, highest reduction was observed in case of LC50 of isolate Bb-01 and LC50 of emamectin, 8.79 (+- 0.35) days, followed by 8.88 (+-0.25) days in combine treatment of LC50 of isolate Bb-01 and LC50 of acetamiprid as compared to all treatments (F=31.29, P <0.0001) (Fig. 1c). In addition, similar trend was observed in case of combine use of Bb-08 and insecticides, where the combine treatment of LC50 of Bb-08 and LC50 of emamectin caused least longevity (11.28 +-0.25) days of females (F=6.37, P <0.0001) (Fig. 1d).

Fecundity and percent hatching

A wide variation was observed in the fecundity among all the treatment levels of fungi and insecticides mixtures (Fig. 2a,b). Significant differences were observed in all treatments especially at highest level of fungal and insecticidal concentrations. Overall, the least number of eggs 113.50 (+-5.72) and 123.00 (+-8.25) were recorded in the treatment with LC50 of isolate Bb-01+ LC50 of emamectin (Fig. 2a) and LC50 of Bb-08+ LC50 of emamectin (Fig. 2b), respectively far <that of other treatments including control.

The results showed wide variation in hatching percentage for all treatments (Fig. 2 c, d). The least egg hatching percentage was recorded in LC50 of Bb-01+LC50 of emamectin (68.80 +- 0.24) (F=4.69, P=0.0001) (Fig. 2c). While, in case of Bb-08, LC50 of isolate Bb-08 +LC50 of lufenuron (70.40 +- 0.59) (F=4.88, P=0.0001) showed maximum reduction in the hatching percentage of eggs of M. domestica (Fig. 2d).

Larval duration, percent pupation, pupal weight and pupal duration

The impact of fungi and insecticides mixtures on larval duration and percent pupation were observed (Table IV). The larval duration ranged from 6.60 to 8.75 days and significant prolongation in larval duration was observed in all treatments. In case of Bb-01, the maximum prolongation 8.56 (+- 0.12) days was observed in treatment with LC50 (Bb-01)+LC50 of lufenuron (F=11.04, P.0001), while in case of Bb-08 maximum prolongation (8.90+-0.26) days was observed in treatment with LC50 (Bb-08)+LC50 of imidacloprid as compared to other treatments including control (F=2.75, P=0.007).

Conversely, a significant decrease in percent pupation was observed for increasing level of fungi and insecticides mixtures. The minimum percent pupation in Bb-01 and insecticide mixtures was recorded in LC50 (Bb-01)+LC50 of emamectin (55.48+-1.53) followed by LC50 (Bb-01)+LC50 of acetamiprid (56.53+-1.53) (F=15.80, P<0.0001). Moreover in case of Bb-08, minimum percent pupation (57.55 +- 0.61) was observed in treatment with LC50 of isolate Bb-08+LC50 of acetamiprid (F=20.30, P<0.0001) as compared to other treatments and control.

The combined effects of fungi and insecticides mixtures caused significant decrease in pupal weight (Table V). In case of combined treatments of Bb-01 and insecticides, the least pupal weight (mg) 10.48 (+- 0.30 mg) was observed in treatment with LC50 (Bb-01)+LC50 of imidacloprid followed by 10.86 (+- 0.51 mg) in case of LC50 (Bb-01)+LC50 of bifenthrin (F=10.53, P<0.0001). In addition, for combined treatments of Bb-08 and insecticides, least pupal weight (11.15 +- 0.35) was observed in treatment containing LC50 (Bb-08)+LC50 of acetamiprid.

In general, a significant prolongation was observed in pupal duration in all treatments. For isolate Bb-01 and insecticides combinations, maximum pupal duration was observed in treatment with LC50 (Bb-01)+LC50 of lufenuron 8.29 (+- 0.11) days (F=5.39, P<0.0001), while for isolate Bb-08, LC50 (Bb-08)+LC50 of emamectin showed maximum prolongation in pupal duration of 7.58 (+- 0.05) days (F=5.53, P<0.0001) (Table V).

Adult emergence and sex ratio

The data showed a significant difference in the adult emergence as result of combined effects of fungi and insecticides mixtures on all treatment levels (Figs. 3a, b). For Bb-01, the lowest adult emergence (60.92+-0.58) was observed in LC50 (Bb-01)+LC50 of imidacloprid, while in case of Bb-08, similar results were observed in treatment with LC50 (Bb-08)+LC50 of imidacloprid (61.25+-0.42).

The combined treatment of fungi and insecticides showed significant difference in sex ratio. For Bb-01, the lowest female ratio was observed in LC50 (Bb-01)+LC50 of bifenthrin (F=1.81., P=0.03) (Table VI). While for Bb-08, lowest percentage of females was found in combined treatment of LC50 of Bb-08+LC50 of emamectin (F=2.21,P=0.02) (Table VI).

DISCUSSION

Insect pathogenic fungi have shown to be an effective biological control agent against M. domestica (Kaufman et al., 2005; Sharififard et al., 2011a). In order to enhance the effectiveness, insect pathogenic fungi can be incorporated with the insecticides doses (Pachamuthu and Kamble, 2000). In our study, concentrations of pathogenic fungi and insecticides that caused 10%, 30% and 50% mortality were measured and these arbitrary concentrations of insecticides and entomopathogenic fungi were considered for further experimentation. Later, these fungal and insecticides concentrations were mixed and tested against M. domestica for the effects on different biological parameters of house fly. Mortality of binary treatments of entomopathogenic fungi and insecticides showed a considerably higher trend than the mortality caused by entomopathogenic fungi and insecticides alone (Tables II, III).

Table II.- Mortality rates of housefly after individual application of fungus and insecticides (LC10, LC30 andLC50).

Name###LC10 (%+-SE)###LC30 (%+-SE)###LC50 (%+-SE)

Bb-01###8.60 +- 0.30d###21.64 +- 0.38c###39.35 +- 0.32e

Bb-08###6.30 +- 0.41e###19.70 +- 0.45d###36.70 +- 0.48f

Acetamiprid###11.30 +- 0.14c###31.40 +- 0.46ab###53.20 +- 0.32b

Bifenthrin###13.30 +- 0.20bc###31.40 +- 0.12ab###51.60 +- 0.25c

Emamectin###15.80 +- 0.25ab###33.61 +- 0.23a###55.40 +- 0.68a

Fipronil###17.6+- 0.48a###34.30 +- 0.25a###56.30+- 0.61a

Imidacloprid###11.62 +- 0.68c###28.53 +- 0.61###49.50 +- 0.47d

Lufenuron###16.17 +- 0.21a###31.60 +- 0.78ab###51.30 +- 0.12c

F-value###34.5###121.3###65.7

P-value###0.02###0.00###0.01

LSD-value###1.35###3.21###2.24

Table III.- Effect of combinations of entomopathogenic fungi and insecticides on percent mortality (+-SE) of M. domestica

###Bb-01###Bb-08

###Expected###Observed###T-###p-###Expected###Observed###T-###P-

###Mfi-Me###Mfi-Me

###mortality###Mortality###test###value###mortality###Mortality###test###value

LC10+LC10

Acetamiprid###18.62+-0.41###30.84+-0.52###2.46###0.032###12.2169###16.32+-0.82###28.14+-0.67###3.45###0.034###11.8169

Bifenthrin###20.13+-0.45###21.55+-1.09###1.1###0.17###1.4189###17.83+-0.21###11.78+-0.63###0.36###0.49###-6.0511

Emamectin###21.90+-1.24###28.54+-1.66###3.1###0.01###6.6364###19.60+-0.76###23.98+-1.24###4.52###0.021###4.3764

Fipronil###23.10+-0.32###17.60+-0.95###1.21###0.21###-5.5024###20.80+-0.51###15.18+-0.30###0.75###0.31###-5.6224

Imidacloprid###18.87+-0.21###21.33+-0.35###1.02###0.01###2.460244###16.57+-0.48###20.48+-0.31###4.22###0.021###3.910244

Lufenuron###22.15+-0.46###34.57+-2.06###2.31###0.012###12.41469###19.86+-0.43###21.53+-0.90###4.31###0.012###1.674689

LC30+LC30

Acetamiprid###43.18+-0.88###56.71+-0.24###4.78###0.042###13.5296###41.24+-0.33###43.26+-1.35###5.43###0.031###2.0196

Bifenthrin###43.18+-1.34###32.33+-1.50###0.42###0.83###-10.8504###41.24+-0.45###21.75+-0.94###0.71###0.94###-19.4904

Emamectin###43.95+-1.78###63.80+-0.90###1.78###0.012###19.84632###42.01+-0.81###42.75+-1.85###2.32###0.041###0.736321

Fipronil###44.18+-2.45###26.24+-1.34###0.61###0.71###-17.9351###42.24+-0.18###22.9+-1.20###0.21###0.74###-19.3351

Imidacloprid###42.03+-1.57###43.01+-1.31###0.82###0.01###0.979609###40.09+-0.51###40.80+-0.96###1.02###0.02###0.709609

Lufenuron###43.25+-0.95###52.68+-1.93###2.51###0.014###9.4256###41.31+-0.32###48.08+-1.52###2.11###0.024###6.7656

LC50+LC50

Acetamiprid###64.25+-2.11###91.43+-0.64###8.31###0.021###27.1824###61.59+-0.54###87.02+-1.08###0.89###0.013###25.4224

Bifenthrin###64.32+-1.56###49.63+-3.06###0.69###0.53###-14.6944###61.67+-0.31###37.76+-3.15###0.71###0.73###-23.9144

Emamectin###64.05+-1.89###87.28+-2.64###4.81###0.032###23.2216###61.41+-0.98###78.33+-1.84###1.53###0.023###16.9216

Fipronil###63.95+-0.93###43.08+-1.54###9.72###0.24###-20.8731###61.30+-1.23###33.10+-0.66###0.13###0.92###-28.2031

Imidacloprid###64.34+-1.03###68.33+-1.01###0.43###0.019###3.9825###61.69+-0.67###62.85+-2.82###1.01###0.021###1.1525

Lufenuron###64.33+-2.45###75.10+-1.40###5.31###0.035###10.7669###61.68+-2.34###67.73+-2.58###1.56###0.035###6.0469

Table VI.- Sublethal effects of entomopathogenic fungi and insecticides mixtures on sex ratio (/(+))(%+- SE) of M. domestica

###(% +- SE)

###Bb-01###Bb-08

###LC10+LC10###LC30+LC30###LC50+LC50###LC10+LC10###LC30+LC30###LC50+LC50

Acetamiprid###49.50 +- 1.94bc###49.25 +- 2.02bc###48.75 +- 1.80bc###50.75 +- 1.84abc###49.75 +- 1.03bcd###48.25 +- 1.60cde

Bifenthrin###52.25 +- 1.80ab###51.00 +- 2.6ab8###46.75 +- 1.03bcd###47.00 +- 0.82cde###51.00 +- 1.78abc###46.50 +- 0.96de

Emamectin###50.75 +- 2.17ab###49.75 +- 2.50 bc###48.00 +- 2.12bc###48.25 +- 1.97 cde###50.00 +- 2.42bcd###45.00 +- 0.58e

Fipronil###51.25 +- 1.65ab###48.05 +- 0.68bc###49.60 +- 1.61bc###48.22 +- 0.48 cde###48.47 +- 1.38 cde###52.82 +- 2.05ab

Imidacloprid###50.20 +- 1.61b###48.00 +- 0.67bc###49.65 +- 1.60bc###47.50 +- 0.69 cde###53.05 +- 1.88a###50.35 +- 2.60bcd

Lufenuron###48.18 +- 0.28cd###51.20 +- 1.47ab###54.75 +- 1.03a###54.25 +- 0.95a###50.35 +- 1.46bcd###48.05 +- 0.68 cde

Control###48.50 +- 0.65cd###48.88 +- 0.66cd###49.25 +- 0.48bc###48.50 +- 0.65 cde###49.35 +- 0.41bcd###48.50 +- 0.65 cde

F###1.81###2.21

P###0.03###0.02

LSD###4.51###4.25

The differences among the efficacy of fungi and insecticides is due to different mode of actions when applied alone. However, the exact mechanisms for effects of fungi and insecticide mixtures are unclear, insecticides may influence the insect cuticle and facilitate penetration for fungal spores, or possibly restrain immune response and facilitate fungal infection process (Hiromori and Nishigaki, 2001). Moreover, insecticides inhibit vegetative growth and spore germination of fungi depending upon nature of active ingredient. In addition, higher toxicity in vitro does not indicate that similar phenomena should occur in field (Archana and Ramaswamy, 2012). Similarly, according to the LC10 ranking the most toxic insecticides are much less efficient when combined with B. bassiana highlights the influence of insecticides on the pathogenic fungi and make it is less compatible as compared to other insecticides, which show more toxicity when used in combination with B. bassiana.

The effectiveness of B. bassiana has been demonstrated for controlling M. domestica. Besides effectiveness of pathogenic fungi against M. domestica, it is important to find approaches that reduce the lethal time by entomopathogenic fungi (Kaufman et al., 2005; Mishra et al., 2011; Sharififard et al., 2011a). The potential use fungi and insecticides in combination have been focused in previous studies (Pachamuthu and Kamble, 2000; Zurek et al., 2002; Jaramillo et al., 2005; Thompson and Brandebburg, 2006; Ericsson et al., 2007; Sharififard et al., 2011b). Most of the studies regarding combined mixtures of insect pathogenic fungi and insecticides were mainly concerned to the mortality of host insect. However, sublethal effects on biological parameters have been overlooked especially for the M. domestica, which may increase the susceptibility of affected insects to other natural enemies.

In addition to direct effect on adult mortality, combined treatments of fungus and insecticide significantly reduced the adult longevity as compared to the average survival time of adult. Due to the reduced life expectancy of the female, the number of eggs were also affected by the treatment. Similar results have been reported for other dipterans where pathogenic fungi resulted in the decreased survival and fecundity with the increasing dose (Flores et al., 2004; Pelizza et al., 2013), while egg hatching percentage was significantly different among all the treatments. In addition to this, similar results were reported by Khodadad et al. (2007) where significant effects of M. anisopliae, B. bassiana and Lecanicillium psalliotae on the percent egg hatchability of Rhipicephalus (Boophilus) annulatu were observed.

Larval duration of M. domestica in the current study was significantly prolonged in all treatments. However, the larval duration was not significantly different at higher doses of fungi and insecticides mixtures. These results are not in accordance with Poprawski et al. (1998) where no significant effect was observed on larval duration of Serangium parcesetosum Sicard. (Coleoptera: Coccinellidae) when tested against entomopathogenic fungi. The results showed significantly different pupation percentage among all treatments as compared to the control. In addition to this, pupal weight was also influenced by the sublethal effects of combined mixtures of fungi and insecticides. Similar kind of results were observed when Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) was investigated for the effect of sublethal concentrations of spinosad, where percentage pupation and pupal weight significantly differed from the control group (Abouelghar et al., 2013; Rehan and Freed, 2015).

The findings of the current research showed a significant prolongation in pupal duration in all treatments as compared to the control which are in accordance to results of Hafez et al. (1997) where pupae of Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae) showed prolonged duration when treated with B. bassiana. These findings are contrary to Poprawski et al. (1998) where no significant differences were observed in pupal duration as compared to the control.

In the current research adult emergence was not significantly affected on all levels of combined mixtures. This is contrary to other research, where adult emergence was observed to be affected in treatments with plant materials for M. domestica and other dipertans (Muse et al., 2003; Khalaf et al., 2009; Elkattan et al., 2011). For sex ratio, significant difference was found only at lower levels of fungus/insecticide mixtures. Disturbance in sex ratio was detected due to mixture of fungi and insecticides as recorded by other studies (Robert and Olson, 1989; Shaalan et al., 2005).

The results of the present study regarding entomopathogenic fungi and insecticides mixtures on biological parameters of M. domestica suggest that the combination of fungi and insecticides prevented the normal developmental stages and duration of M. domestica. The dosage of synthetic insecticides can act as physiological stressors and/or behavioral modifiers, thereby predisposing insects to diseases (Inglis et al., 2001). The combined mixtures of isolate Bb-01 with acetamiprid, emamectin, imidacloprid, or lufenuron significantly altered the normal development of M. domestica. Integrating entomopathogenic fungi with insecticides may have advantages. This approach will not only increase mortality in pest and possibility can reduce the time to kill.

CONCLUSION

In conclusion, the combined use of insect pathogenic fungi and a chemical insecticide may be an important component of integrated pest management of M. domestica, which needs to be tested under field conditions to determine its efficacy.

Statement of conflict of interest

Authors have declared no conflict of interest.

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Author:Farooq, Muzammil; Freed, Shoaib
Publication:Pakistan Journal of Zoology
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Date:Oct 31, 2016
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