Assessment of Binary Mixtures of Entomopathogenic Fungi and Chemical Insecticides on Biological Parameters of Culex pipiens (Diptera: Culicidae) under Laboratory and Field Conditions

Rana Fartab Shoukat1, Shoaib Freed1,*, Kanwar Waqas Ahmad1 and Ateeq-ur-Rehman2

1Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan

2Department of Plant Pathology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan

ABSTRACT

Southern house mosquito, Culex pipiens (Diptera: Culicidae), is most common agent for transferring a mass of pathogens i.e., West Nile Virus (WNV), avian pox virus (APV) etc. in rural and urban areas. To mitigate these problems study was conducted by using binary mixtures of entomopathogenic fungi, Beauveria bassiana (isolates Bb-01, Bb-10), Metarhizium anisopliae var. anisopliae (isolate Ma-11.1, Ma-2.4) and Isaria fumosorosea (isolates If-2.3, If-02) and chemical insecticides i.e., bifenthrin, lambda cyhalothrin, imidacloprid, triazophos, spinosad, pyriproxyfen and nitrinpyrum, as larvicides against C. pipiens. Highest larval percent mortality (73.3 ± 4.7) was observed after application of Ma-11.1(LC40) + nitenpyram (LC40) mixtures under laboratory and field conditions (71.5 ± 7.4). The results showed a significant effect of binary treatments of fungi and chemical insecticides on biological parameters of C. pipiens and its progeny (P<0.05). The insect pathogenic fungi showed compatibility with insecticides and the combined application can improve the management program of C. pipiens.

Article Information

Received 12 June 2016

Revised 16 January 2017

Accepted 27 July 2017

Available online 12 January 2018

Authors’ Contribution

RFS and KWA performed experiment, analysed the data and wrote the manuscript. SF provided technical assistance, supervision and helped in writing the manuscript. AR compiled the manuscript.

Key words

Culex pipiens, Mosquito, Larvicides, Biological parameters, Entomopathogenic fungi, Progeny.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.1.299.309

* Corresponding author: [email protected]

0030-9923/2018/0001-0299 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

Culex pipiens (Diptera: Culicidae), is the most common mosquito in rural and urban areas which in capable of transferring a number of pathogens i.e., West Nile Virus (WNV) (CDC, 2002; Turell et al., 2001; Alouani et al., 2009), avian pox virus, bird malaria pathogen, dog heartworm, Dirofilaria immitis, filarial nematode, Wuchereria bancrofti (Vinogradova, 2000) and St. Louis encephalitis (Eldridge et al., 2000). One million people around the world are suffered annually from mosquito transmitted diseases (Kager, 2002). Mostly, synthetic pyrethroids, organophosphates and insect growth regulators are used for the control of adults and larvae of mosquito (Zaim et al., 2000; Hougard et al., 2002; Cui et al., 2006; WHO, 2006).

Over the last five decades relatively large number of problems have been raised due to the misuse of chemical insecticides namely, insecticide resistance, environmental and water pollution, toxic hazards to humans and other non-target organisms (Jirakanjanakit et al., 2007; Seccacini et al., 2008; Al-Sarar, 2010; Khandagle et al., 2011). These problems require the development of effective, easy to apply and cheap substitutes by using eco-friendly products to control the mosquitoes (Liu et al., 2004, 2013; Nielsen and Lewis, 2012). In order to mitigate these problems, a major emphasis has recently been given by using insect pathogenic fungi (IPF) as larvicides (Howard et al., 2010; Mahesh et al., 2012). In general, mosquitoes show susceptibility towards pathogenic fungi and its derived products. The pathogenic fungi being low toxic to non-target organisms and its activity as larvicides proves to be a promising approach for the biological control of insect pests (Soni and Soam, 2010; Bukhari et al., 2011; Freed et al., 2012). In addition to this, entomopathogenic microorganisms have the advantage to be generally specific without affecting other natural enemies as compared to most of chemical insecticides (Perinotto et al., 2012).

Beauveria bassiana and Metarhizium anisopliae var. anisopliae are two of the most common cosmopolitan fungi which are pathogenic to a number of insect pests (Nussenbaum and Lecuona, 2012). The objective of current study was to investigate the efficiency of individual and combined application of fungi and insecticides for developing an eco-friendly approach and to investigate the effect of sub-lethal doses on the biological parameters of C. pipiens.

Materials and Methods

Mosquito collection and rearing

Larval collections (all instars) were carried out from Multan, Punjab, Pakistan from different sites and were pooled per locality. Larvae were brought to the Laboratory of Insect Microbiology at B.Z. University (BZU) Multan, where these were reared under standard controlled conditions (26±2°C, 80±10% relative humidity (RH) and 12:12 L-D photoperiod). Larvae were fed daily with baby fish food. The adult emergence was morphologically identified using identification keys and shifted in disinfected plastic cages (1.5 × 2 ft). Adult males were feed on 10% sugar solution, while females were given blood meal by feeding on laboratory white albumen mice. Female mosquitoes laid eggs in the form of patches in glass petri dishes containing 10ml water placed in plastic cages. Larvae on hatching were shifted in glass jars (10 ×15cm) having 500 ml tap water and were feed on fish food till pupation. Water and diet of each jar was changed after every 48 h.

Different isolates of B. bassiana (Bb-01, Bb-10), M. anisopliae var. anisopliae (Ma-11.1, Ma-2.4) and I. fumosorosea (If-2.3, If-02) (Table I) already present in the Laboratory of Insect Microbiology, BZU, Multan were used. Monoconidial cultures of the isolates grown on PDA were used. For further propagation rice media was used. Stock solutions were made and stored at 4°C for further use.

Commercial formulations of different chemical insecticides were purchased for bioassay from the pesticide market of Multan, Pakistan (Table I).

Preparation of concentrations of fungi and insecticides

The spore concentrations of entomopathogenic fungi were measured using the haemocytometer and the desired concentrations (4×108, 3×108, 2×108, 1×108 and 1×107 spores/ml) and (0.005, 0.0025, 0.00125, 0.000625, 0.00031 ppm) of fungi and insecticides were prepared for bioassay. On the other hand, the binary mixtures were made by using the following scheme e.g., fungi (LC40) + insecticide (LC40), while similar scheme was adopted for other concentrations.

Table I.- List of the isolates of entomopathogenic fungi and insecticides used in the experiments.

The experiment was conducted under the Completely Randomized Design (CRD) with four replications in each treatment. All fungi and insecticides were screened to calculate LC50, LC40, LC30, LC20 and LC10 values for both laboratory and field experiments. Three fungal isolates from the above mentioned fungi were selected with lowest LC50 values. Mixtures of different concentrations of fungi + insecticides was applied on both laboratory and field populations. Small plastic containers with a capacity of 450 ml water were used for experimentation. In each container, 250 ml serially diluted suspension was poured and 15 larvae of same age belonging to late 3rd instar were released and provided with sufficient fish food. The containers were labelled and placed under laboratory condition (temperature, 25±1°C, RH. 75±2% and Photo period 10L-14D h) in Laboratory of Insect Microbiology, BZU, Multan, while for field experiments; these were placed under shady places (Temperature, ≈ 29±2°C, RH. 55±2%) in fields of BZU, Multan. Data of mortality was taken for seven days in case of fungi (Khan et al., 2014), three days in case of insecticides and seven days in case of binary mixtures. The data of precent pupation, pupal duration, precent emergence and sex ratio was recorded from beginning to end of experiment (Sivagnaname and Kalyanasundaram, 2004). LC50, LC40, LC30, LC20 and LC10 values of all fungi and insecticides were calculated by using POLO-PC software (LeOra Software, 2003). The means were analyzed by analytical software (Statistix version 8.1) and compared by LSD test at 0.05 probability levels.

Results

Pre-experimentation was conducted on laboratory and field populations of C. pipiens for determining LC50, LC40, LC30, LC20 and LC10 values of six isolates of fungi and seven chemical insecticides. Later three fungi were selected on the basis of least LC50 values and sub-lethal doses were used in binary mixtures to check the mortality and after effects on the progeny (Table II).

Larval mortality after fungi + insecticide treatment

The mortality of C. pipiens in laboratory population after the application of fungi + insecticide is shown in Table III. Highest percent mortality (73.3 ± 4.7) was observed when mixture containing Ma-11.1 (LC40) + spinosad (LC40) was applied followed by (71.7 ± 4.1) with the application of Ma-11.1 (LC40) + nitenpyram (LC40). The results showed a concentration dependent response (F=61.65, P=0.0087, df=6), which increased with the increase in concentration. On the other hand, the highest percent mortality (71.5 ± 7.4) was recorded after the application of mixture containing Ma-11.1(LC40) + spinosad (LC40) (F=176, P=0.0023, df=6) under field conditions (Table III).

Table II.- Calculated doses of fungi (spores/ml) and insecticides (ppm) for binary treatment on C. pipiens (laboratory and field trials).

Table III.- Larval mortality (%) of C. pipiens in laboratory and field trials as a result of binary treatment of fungi and insecticides.

Table V.- Pupal duration of C. pipiens under laboratory and field trials as a result of combined application of fungi and insecticides.

Least pupation (16.3 ± 0.8) was recorded after the application of Ma-11.1+ spinosad (LC40) followed by Ma-11.1 + nitenpyram (LC40) which caused (18.3 ± 0.4) percent pupation (F=65, P=0.008, df = 6) (Table IV). On the other hand, least percent pupation (34.2 ± 0.9) after treatment of mixture (fungi + insecticides) was recorded after treatment with Ma-11.1+ nitenpyram (LC40) mixture, followed by Bb-01 + triazophos (LC40) causing (38.0 ± 0.3) precent mortality (F=109, P=0.0001, df=6) (Table IV).

The results depicted longest pupal duration after the application of higher concentrations of fungi + insecticide on laboratory population (Table V). Longest pupal duration (11.0 ± 0.3) days was recorded when mixture of Bb-01 + nitenpyram (LC40) followed Ma-11.1+ nitenpyram (LC40) (10.1 ± 0.7) days (F=56, P=0.002, df = 6). In contrast to this the binary mixtures application of fungi and insecticides under field condition showed longest pupal duration (10.0±0.3) (days) after treatment with Bb-01 + nitenpyram (LC40) (F=151, P=0.008, df = 6) as compared to the control.

Emergence after fungi + insecticides treatment

Lowest adult emergence (10.8 ± 0.6) was recorded after treatment with Ma-11.1+ nitenpyram (LC40) followed by (13.2 ± 0.9) treated with Ma-11.1+ spinosad (LC40) (F=158, P=0.001, df=6) (Table VI). Conversely lowest precent emergence (19.1 ± 0.4) was observed after application of Bb-01 + nitenpyram (LC40) (F=136, P=0.0076, df=6) under field conditions, while maximum precent emergence (97.9 ± 0.3) was recorded in the control treatment. The data regarding sex ratio after application of fungi and insecticides mixtures showed non-significant difference on all levels of treatments.

Discussion

The current study was conducted to test the effectiveness of different insect pathogenic fungi and insecticides individually and in combinations against larval stages of C. pipiens. Our previous research has depicted the compatibility of these fungi with the insecticides (Akbar et al., 2012). The previous research has shown the effectiveness of entomopathogenic fungi against dengue mosquitoes (Scholte et al., 2007; Paula et al., 2008), while M. anisopliae and I. fumosorosea have been used as virulent fungi for the control of different insect pests of various crops (Kaufman et al., 2005; Sharififard et al., 2011; Mishra et al., 2011). In addition to this various entomopathogenic fungi in combination with insecticides have been used for the management of a number of insect pests in past (Sharififard et al., 2011; Archana and Ramaswamy, 2012; Kassab et al., 2014).

Present study shows the combined effect of entomopathogenic fungi and synthetic insecticides on mortality and different biological parameters of C. pipiens. Highest larval mortality i.e., 73.3 and 71.5% in laboratory and field trials was recorded after the combined treatment of Ma-11.1 with spinosad (LC40), respectively which lies in favour of Anderson et al. (1989) who reported rapid mortality of Colorado potato beetle in laboratory and field trials due to combined application of B. bassiana with different insecticides. Similar results have been reported by Flores et al. (2004) and Pelizza et al. (2013) which depict decreased survival rate of Aedes aegypti after application of binary mixtures of entomopathogenic fungi and insecticides.

Percent pupation of C. pipiens was concentration dependent, higher concentrations of binary mixtures of entomopathogenic fungi and insecticides resulted in low percent pupation which lies in favor of Prasad and Veerwal (2012) who reported same results after application of B. bassiana on Anopheles stephensi. Pupal duration seemed to be prolonged after application of entomopathogenic fungi and insecticides in laboratory and field trails after application of binary mixtures (Bb-01 + nitenpyram LC40). The results are in accordance to findings of Malarvannan et al. (2010) who stated increased pupal duration of Spodoptera litura Fabricius (Lepidoptera: Noctuidae) after treatment of entomopathogenic fungi.

Percent emergence of C. pipiens varied with the concentration of mixtures in field and laboratory trails, which relates with the findings of El-Razik et al. (2013) who observed the decrease in the adult development after treatment of binary mixtures (fungi + insecticides) on Callosobruchus maculatus (F). On the other hand, the data regarding sex ratio of C. pipiens showed non-significant differences after treatment of mixture of fungi and insecticides both in laboratory and field trails. Similar results were observed by Shaalan et al. (2005) who reported abnormal sex ratio in A. aegypti after application of mixtures of fungi and insecticides. Combination of entomopathogenic fungi and insecticides showed potential to be used in mixtures to counter insecticide resistance (Farenhorsta et al., 2009). Binary application of insecticide and fungi cause stress and disease for better management of C. pipiens. In conclusion mixture of entomopathogenic fungi and insecticides could be used as an effective tool for integrated management of C. pipiens.

Acknowledgements

The authors thank editor and anonymous referees for their invaluable comments and suggestions.

Statement of conflict of interest

Authors have declared no conflict of interest.

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