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Toxins of bacillus thuringiensis var. israelensis for control of malaria vector Anopheles stephensi under laboratory and semi field conditions.

Byline: Sarwat Naz Azhar Maqbool Mansoor Ud Din Ahmad and Aftab Ahmad Anjum

Abstract

Two commercial preparations of Bacillus thuringiensis var. israelensis VectoBac WDG and Mousticide WP were evaluated and compared for larval control of Anopheles stephensi a malaria vector in Pakistan under laboratory and semi field conditions. An. stephensi larvae were susceptible to VectoBac WDG and Mousticide WP but susceptibility was higher for VectoBac WDG. In semi field experiment VectoBac WDG @ 0.4 mg/L (1X LC100) and1.6 mg/L (4X LC100) remained more effective against An. stephensi L3 larvae up to day 7 whereas larval mortality dropped below 50% at day 14 of post VectoBac WDG application. Mousticide WP @ 1.22 mg/L (1X LC100) produced 79.33% mean mortality of An. stephensi L3 at 1 day post treatment. The larval mortality dropped to 30% at day 14 of post treatment with Mousticide WP. No significant difference was seen in larval mortality between two dose rates of VectoBac WDG and Mousticide WP. Copyright 2014 Friends Science Publishers

Keywords: An. stephensi; Bacillus thuringiensis var. israelensis; Bio larvicides

Introduction

Malaria is a mosquito-borne protozoan parasitic disease which badly affects almost half a billion people living in109 countries in Africa Asia and Latin America (WHO2010). Despite of known gross under-reporting of malaria in Pakistan the estimated burden of malaria is 1.5 million cases per year (Murtaza et al. 2009). 140 million people in the country are at risk of malaria and 18% of the total population is living in high risk situations (WHO 2009).Malaria is predominantly more prevalent in the rural areas(Mukhtar 2004).Among 22 Anopheles species two sub-species and one variety reported from Pakistan only two species Anopheles culicifacies and Anopheles stephensi are known vectors of malaria. An. culicifacies is a confirmed primary vector of malaria in rural areas (Hick and Majid 1937; Pervez and Shah 1989; Herrel et al. 2004). An. stephensi has been generally considered to be a secondary vector of malaria (Rehman and Muttalib 1967). However recent evidences from rural areas of Punjab suggest that it may be playing an important role in transmission of malaria in rural areas as well (Kakar et al. 2010).Vector control with residual indoor spraying and insecticide treated bed nets are effective approaches to combat malaria. Due to rise and spread of resistance to residual insecticides in mosquito vectors (Hemingway et al.2002; WHO 2004) the reliance on adulticides as sole tool may be inappropriate for control of mosquitoes in many situations. The treatment of mosquito breeding places with microbiolarvicides is good option to reduce the vector burdens thus resulting a decrease in malaria transmission (Killeen et al. 2002; Majambere et al. 2007).Bacillus thuringiensis var. Israelensis (Bti) a gram positive entomopathogen bacteria has rapid larvicidal activity against black flies and mosquitoes and due to its environmental safety and specificity to nematoceran Diptera especially mosquitoes (Aly et al. 1987; Fillinger et al.2003). The larvicidal potentials of Bti have been recognized since 1977 (Goldberg and Margalit 1977) and Bti has become mosquito control agents of choice almost throughout the world.Resistance in vectors of malaria against different classes of insecticides has been reported from Pakistan (Rathor et al. 1985). But studies on complementary/ alternative method like B. thuringiensis var. Israelensis (Bti) or other biological control agents against vectors of malaria in the country are rather limited (Jahan and Hussain 2011).Present study was designed to determine and compare the efficacy and residual effect of two commercial preparations of microbial larvicide B. thuringiensis var. Israelensis (Bti) against larvae of malaria vector An. stephensi.Materials and Methods

Larvicidings

Two commercial preparations of bio larvicides with trade names viz.1) VectoBac WDG: water dispersible granules of B. thuringiensis var. Israelensis strain AM65-52 3000ITU/mg from (Valent biosciences USA).2) Mousticide WP: a wettable powder of B. thuringiensis var. Israelensis strain H14 1410 ITU/mg with 20% TNOF- Yeast (Ento Genex Malaysia) were used.

Mosquito Larvae Source

Bio assays were conducted with laboratory reared larvae originally derived from wild-caught An. stephensi mosquitoes.For rearing the colony of wild An. stephensi hand collection of indoor resting An. stephensi mosquitoes was carried out by using suction tube and torch-light. Mosquitoes were held in 30cm3 plastic cages and were provided cotton wool pads soaked in 20% sucrose. Mosquitoes were reared at 273C 7010% relative humidity and a 16L: 8D photo-period Female were fed on restricted albino mice for 30minutes two times per week. After 72 h of blood feeding engorged females were set foregg laying in cages having container filled halfway with non-chlorinated drinking water and lined with paper towel for oviposition. Eggs on paper strips were removed dried for one day and then stored in a zip lock bag with wet paper towel to maintain high humidity. Eggs were placed in plastic cups for hatching into first instar larvae. A pinch of fish food was added to cups and held overnight until larvae were large enough to sort in trays. These larvae were transferred to disposable plastic trays (35 cm A- 30 cm A- 5 cm) filled with 1200 mL of distilled water. Larvae were reared at a fixed density of 200 larvae per tray to reduce variation in adult size at emergence. Larvae were fed with 2 drops of 10% sugar solution for 3 days and then a pinch of liver powder was spread on the surface of water twice daily. On 11 to 15 days post hatching pupae were collected in plastic cups containing distilled water and placed in emergence cages. Adult emerging within 24 h were provided with 10% glucose solution. This rearing and maintenance regime was adopted throughout the study period.

Laboratory Bio Assays

Bio assays were conducted following the guide line ofWorld Health Organization for testing larval susceptibilityto larvicides with some modification as described by Brownet al. (2000).Laboratory bio assays were carried in 120 mLdisposable plastic cups each holding 100 mL of tap water (non-chlorinated). To each cup 3rd instar larvae of An. stephensi (25 No.) were introduced and left for acclimatization for 2-3 h. Laboratory bio assays were conducted with 5 to 6 concentrations from 0.01-2 ppm (Becker and Bozhao 1989). First 1% stock suspensions of VectoBac WDG and Mousticide were made and calculated amounts of larvicide suspensions were transferred to test cups to achieve the required concentrations. Test and control cups were kept at an ambient temperature (26-30C) and humidity 80% for 24 h. After 24 h exposure death or the lack of reaction to gentle prodding with a glass pipette were observed and observations were recorded. No food was added to the test and control bioassay cups. Each concentration was replicated thrice and three untreated cups were used as control. During whole experiment if larval mortality in control container exceeded 10% the test was discarded and repeated. Each formulation was tested on three different occasions. The stock suspensions and their dilutions were freshly prepared on each occasion.

Determination of Residual Effect of Bti larvicides underSemi Field Conditions

Trials regarding evaluation of residual effect of two microbial insecticides viz. VectoBac(R) WDG and Mousticide were conducted from July to October 2011. Fifteen plastic buckets (each with a diameter of 30 cm) were filled with 10 L of water collected from irrigation channel. All the 15 buckets were evenly distributed in five groups (A-E) and placed outdoors in sunlight at least 48 h prior to the experiment. Twelve buckets in groups A B C and D were treated with the test products and three buckets (in group E) with no treatment served as controls. VectoBac(R) WDG was applied @ 1X LC100 to buckets in group A and VectoBac(R) WDG @ 4X LC100 to buckets in group B. Mousticide was applied @ 1X LC100 to buckets in group C and @ 4X LC100 to buckets in group D. Each test product was applied once to each test bucket at the start of experiment. Thirty third-instar larvae of An. stephensi in a cage (locally developed which allowed free movement of water inside) were introduced into each bucket on day1 day7 day 14 day 21 day 28 post treatment or until the efficacy was less than 50%. First evaluations of larvae were carried out after 24 h of introduction into containers. Larval mortality was recorded daily up to 48 h at each introduction period. The experiment was repeated on three different times.

Statistical Analysis

To estimate the dosage response of exposed larvae Probit regression analysis was used: LC

LC

at 95% confidence

limits of each lethal level and slope values were determined. To determine possible effect of time periods on the efficacy of two larvicides under semi field conditions mean larval mortalities were compared using analysis of variance

(ANOVA) and multiple comparisons were done using LSD technique. All analyses were carried out using Microsoft Excel 2007 and statistical software SPSS version 16 (SPSS Inc Chicago IL USA).

Results

Susceptibility of An. stephensi Larvae to Commercial Preparation of Bacillus thuringiensis var. Israelensis (Bti) Larvicides.

Residual effect of VectoBac WDG and Mousticide WP

In semi field experiment VectoBac WDG @ 0.4 mg/L (1X LC100) performed effectively against An. stephensi L3 larvae up to day 7 with 98.660.84 larval mortality whereas at day35 the lowest larval mortality was 14.170.70. The larvalmortality dropped below 50% at day 14 of post VectoBac WDG @ 0.4 mg/L (1X LC100) treatment (Table 2). The mean mortalitySE of An. stephensi L3 larvae at day171428 and 35 of post VectoBac WDG @ 1.6 mg/L (4X LC100) treatment are shown in (Table 2). Post Hoc test (LSD) showed that mean mortalitySE between two treatment dosages of VectoBac WDG 0.4 mg/L (1X LC100) and 1.6 mg/L (4X LC100) differed insignificantly at day171428 and 35 of post treatments (Pgreater than 0.05) (Table 3).Mousticide WP @ 1.22 mg/L (1X LC100) produced mean mortalitySE of An. stephensi L3 at 1 day post treatment was 79.33 0.67 whereas at day 28 post treatment the larval mortality was 07.830.40. The larval mortality dropped below 50% at day 14 of post treatment with Mousticide WP @ 1.22 mg/L (1X LC100) (Table 2). The mean mortalitySE of An. stephensi L3 larvae at day171428 and 35 of Mousticide WP @ 4.88 mg/L (4XLC100) post treatment are shown in (Table 2). Post Hoc test (LSD) showed that mean mortalitySE between two treatment dosages of Mousticide WP 1.22 mg/L (1X LC100) and 4.88 mg/L (4X LC100) differed insignificantly at day171428 and 35 of post treatments (Pgreater than 0.05) (Table 3).

Discussion

Bacillus thuringiensis var. Israelensis (Bti) has been recognized as an efficient biolarvicide against many malaria vector species (Lacey and Lacey 1990; Becker and Margalit 1993; Das and Amalraj 1997; Fillinger and Lindsay 2006; Mwangangi et al. 2011). In Pakistan Bti mosquito larvicides are not available locally. So published studies on laboratory and field trials of Bti against vectors of malaria and other mosquito species in Pakistan are quite limited (Rathor et al. 1985).In this study larvae of An. stephensi were found almost highly susceptible to Bti formulations VectoBac WDG and Mousticide WP under laboratory conditions. Parallel to present study are findings of Jahan and Hussain(2011) who reported that 0.11 ppm of technical powder ofBti (VectoBac TP) containing 5000 ITU/mg caused 95%mortality in 3

stage larvae of An. stephensi after 24 hours

exposure.Rathor et al. (1985) in Pakistan using an aqueoussuspension of Bti (ABG-6145) containing 587 ITU Bti/mghad reported complete mortality of the larvae of An.stephensi at 1 ppm dosage.The level of larvicidal and hence the mortality encountered was different for VectoBac WDG and Mousticide WP. The doses of VectoBac WDG caused 50% and 95% mortality of 3rd instars of An. stephensi in this study were consistent with LC50 LC95 of Bti formulations reported by Fillinger et al. (2003); Majambere et al. (2007) against 3rd instars of African Anopheles vectors of malaria. However the doses of Mousticide TP caused 50% and 95% mortality of 3rd instars of An. stephensi was higher than the doses recorded for VectoBac WDG. It could be attributed to potency of Mousticide TP which contained less toxic units of Bti than VectoBac WDG. However the efficacy of a Bti may also be affected by physical nature release mechanism of active ingredient and settling rate of the formulation (Lacoursiere and Charpentier 1988). For VectoBac WDG formulation the crystals of Bti form deposits slowly towards the bottoms of the lodging than Mousticide TP formulation and provide sufficient quantity of Bti crystals to Anopheles larvae before their sedimentation. Larvae of Anopheles would show higher death rate if crystals of Bti are delivered under a floating formulation (Aly et al. 1987).Laboratory trials show a residual effect of Bti for 100 days (Ignoffo et al. 1981) but adequate formulations that display long term persistence in the field are not yet available (Kramer 1990; Skovmand and Sanogo 1999). In this study VectoBac WDG caused 99% mortality of An. stephensi larvae up to 7day of post application and Mousticide WP showed maximum mortality 80% by 1day post application. The findings of this study are more or less similar to the studies conducted in other parts of the world. Barbazan et al. (1998); Fillinger and Lindsay (2006); Kahindi et al. (2008); Mwangangi et al. (2011) reported complete reduction of Anopheles larvae by 6 day post treatment of Bti in open field trials in East African region.A number of environmental factors such as direct exposure to increased sunlight and high atmospheric temperature influence Bti effectiveness in the field (Ignoffo

Table 1: Probit analysis of Bti VectoBac WDG (strain AM65-52 3000 1TU/mg) and Bti Mousticide WP (strain H14 1410 IU ) against 3 instar larvae of An. stephensi for 24 h exposure time

Bti formulations###LC50 95%CL###LC95 95%CL###SlopSE

VectoBac WDG (AM65-52 3000 1TU/mg)###0.046; (0.038-0.054)###0.149; (0.112-0.243)###3.213.465###1.638

Mousticide WP; (H141200 ITU/mg + TMOF)###0.203; (0.170-0.242)###0.564; (0.439-0.822)###3.710.464###3.786

Table 2: Residual effect of VectoBac WDG and Mousticide WP at two treatment dosages under semi field conditions

Days###Post###Mean Mortality %age of An. stephensi SE

Treatment###Control###VectoBac WDG 0.4 mg/L###VectoBac WDG 1.6 mg/L Mousticide WP 1.22 mg/L###Mousticide WP 4.88 mg/L

1###2.000.89###99.230.66###100.000.00###79.330.66###80.001.03

7###0.660.66###98.660.84###99.330.66###61.332.23###62.662.45

14###0.330.14###47.330.66###46.660.84###30.001.36###30.331.30

21###1.330.84###40.001.03###40.331.08###21.330.84###22.160.98

28###2.781.33###20.000.63###20.500.92###7.830.40###7.830.40

35###1.830.43###14.160.70###14.330.84###0.500.34###0.500.34

Table 3: Pair Wise Comparison of Treatments

Days post treatment###Larvicide###Control###VectoBac 0.4 mg/L###VectoBac 1.6 mg/L###Mousticide 1.22 mg/L###Mousticide 4.88 mg/L

1###VectoBac 0.4mg/L###0.000###-###0.531###0.000###0.000

###VectoBac 1.6mg/L###0.000###0.531###-###0.000###0.000

###Mousticide 1.22mg/L###0.000###0.000###0.000###-###0.531

###Mousticide 4.88mg/L###0.000###0.000###0.000###0.531###-

7###VectoBac 0.4mg/L###0.000###-###0.769###0.000###0.000

###VectoBac 1.6mg/L###0.000###0.769###-###0.000###0.000

###Mousticide 1.22mg/L###0.000###0.000###0.000###-###0.558

###Mousticide 4.88mg/L###0.000###0.000###0.000###0.558###-

14###VectoBac 0.4mg/L###0.000###-###0.655###0.000###0.000

###VectoBac 1.6mg/L###0.000###0.655###-###0.000###0.000

###Mousticide 1.22mg/L###0.000###0.000###0.000###-###0.808

###Mousticide 4.88mg/L###0.000###0.000###0.000###0.808###-

21###VectoBac 0.4mg/L###0.000###-###0.808###0.000###0.000

###VectoBac 1.6mg/L###0.000###0.808###-###0.000###0.000

###Mousticide 1.22mg/L###0.000###0.000###0.000###-###0.556

###Mousticide 4.88mg/L###0.000###0.000###0.000###0.556###-

28###VectoBac 0.4mg/L###0.000###-###0.655###0.000###0.000

###VectoBac 1.6mg/L###0.000###0.655###-###0.000###0.000

###Mousticide 1.22mg/L###0.000###0.000###0.000###-###1.000

###Mousticide 4.88mg/L###0.000###0.000###0.000###1.000###-

35###VectoBac 0.4mg/L###0.000###-###0.884###0.000###0.000

###VectoBac 1.6mg/L###0.000###0.884###-###0.000###0.000

###Mousticide 1.22mg/L###0.469###0.000###0.000###-###1.000

###Mousticide 4.88mg/L###0.469###0.000###0.000###1.000###-

et al. 1981; Kramer 1990; Becker et al. 1992). The climate conditions of the study area may have contributed to lower persistence because containers were placed outdoors. Intensity of sunlight as well as the water temperature was high during the study period it is most probably that the combined effect of higher intensity of sunlight and high temperature reduced the potency of Bti formulations. Becker and Margalit (1993) found that sunlight reduced the effectiveness of Bti approximately four fold against Cx. pipiens in sunlit sites than shaded sites. Similarly to other authors in this study higher concentrations of Bti formulations failed to enhance the residual activity (Karch et al. 1991; Gelernter and Schwab 1993; Kroeger et al. 1995; Fillinger et al. 2003).In conclusion it is evident that the Bti offers a goodpotential for larval control of local strain of An. stephensi under laboratory conditions. Under semi field conditions the floating preparation of Bti VectoBac(R) WDG remained toxic to larvae of An. stephensi for longer duration than wettable powder preparation Mousticide.

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Author:Naz, Sarwat; Maqbool, Azhar; Ahmad, Mansoor Ud Din; Anjum, Aftab Ahmad
Publication:International Journal of Agriculture and Biology
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Date:Oct 31, 2014
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