Journal of Asia-Pacific Entomology 17 (2014) 175–181

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Journal of Asia-Pacific Entomology

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Effects of NeemAzal on marker enzymes and hemocyte phagocyticactivity of larvae and pupae of the vector mosquito Aedes aegypti

Arunagirinathan Koodalingam ⁎, Ramadoss Deepalakshmi, Manoharan Ammu, Arumugam RajalakshmiDepartment of Biotechnology, Sri Sankara Arts and Science College, Enathur, Kanchipuram, 631 561 Tamil Nadu, India

⁎ Corresponding author. Tel.: +91 44 27264066x19.E-mail address: akoodalingam@gmail.com (A. Koodali

1226-8615/$ – see front matter © 2014 Korean SocietyAll rights reserved.http://dx.doi.org/10.1016/j.aspen.2013.12.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 May 2013Revised 29 December 2013Accepted 31 December 2013

Keywords:AzadirachtinNeemMosquito controlEsterasesPhosphatases

Many of the neem based botanical biocides are currently studied to a greater extent because of the possibility oftheir use in eco-friendly control of pests and vectors. However, no report was available to assess the impact ofneem based formulation, NeemAzal on marker enzymes and hemocyte mediated cellular immune responsesof important vector mosquito A. aegypti. The NeemAzal found to exert larvicidal and pupicidal activities againstA. aegypti developmental stages. The pupae appear to be more susceptible to the treatment. Further, a significantincrease in the level of total protein (31%), α-carboxylesterase (121%), β-carboxylesterase (46%), acid phosphatase(62%) and alkaline phosphatase (37%)was observed in larvae upon exposure toNeemAzal.Moreover, treated pupaeshowed increased level of acetylcholinesterase (116%) and acid phosphatase (43%) while α-carboxylesterase(34%), β-carboxylesterase (12%) levels were simultaneously decreased, and no significant changes in alkaline phos-phatase were noticed. Qualitative analysis also revealed that the exposure considerably modulated the larvalβ-carboxylesterase isoenzyme profile whereas little changes were noticed on phosphatases. On the other handhemocyte viability of larvae (18%) andpupae (16%) aswell as phagocytic ability of larval (48%) andpupal hemocytes(44%) against yeast target was significantly reduced upon NeemAzal exposure. We demonstrated for the first timethat the NeemAzal differentially affected the marker enzymes and created immuno-suppressive state by reducingthe phagocytic ability of hemocytes of larvae and pupae of A. aegypti.© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection

Society. Published by Elsevier B.V. All rights reserved.

Introduction

The occurrence of arthropod-borne diseases continues to posesignificant health risk in many parts of the world. In recent years,mosquito-borne diseases have emerged as a serious public health prob-lem in terms of morbidity, mortality and economic costs in countries ofthe South-East Asia region, including India (Vijayakumar et al., 2005;Garg et al., 2008). Among various mosquito-borne diseases, denguefever, Japanese encephalitis and malaria occur world-wide in epidemicform almost on an annual basis (Tolle, 2009), and they globally threaten2.5 billion people at any time point (WHO, 2013). In India, the recentoutbreak of dengue fever transmitted by Aedes aegyptiwith an alarmingreport of 50,000 cases in 2012 stunned policymakers, thereby imposingurgent need for development of control measures (Garg et al., 2008;Raheel et al., 2011; Chaudhuri, 2013). At present, there is no specifictreatment or vaccine available for dengue, and vector control is, there-fore, the only availablemeans of prevention of this deadly disease. How-ever, the success of this approach appears to be limited, because offrequent reports of A. aegypti resistance to common class of insecticidesincluding organochlorines, organophosphates, carbamates and pyre-throids (Hemingway and Ranson, 2000; Singh et al., 2011).

ngam).

of Applied Entomology, Taiwan Ent

To overcome the known drawbacks of synthetic chemical insecti-cides, and the increasing cost of introduction of new chemical insecti-cides into the market, the focus is shifted towards alternative controlmethods using biopesticides (Casida and Quistad, 1998; Cantrell et al.,2012). The eco-friendly neem and Bacillus thuringiensis (Bt)-basedproducts with potent antimosquito property currently favoured in thevector control programme all over the world, because of their effective-ness against broad range of insects, low environmental impact and lesschance for development of resistance (Hall andMenn, 1998; Singh et al.,2011). However, certain disadvantages in applications of Bt-based prod-ucts have been reported,which include limitedfield stability, ineffectiveagainst certain insects, and finally it also allows development of resis-tance in target insects (Chevillon et al., 2001; Ferré and Van Rie, 2002;Paris et al., 2011). Due to these undesirable features, usages of Bt-based products are not the most preferred commercial insecticides(Ferré and Van Rie, 2002). Alternatively, insecticidal botanicals aregaining more attention in vector control programme due to their eco-friendly nature and less chances for the development of resistance(Isman, 2006).

Azadirachtin is one of the most studied botanical insecticides fromneem tree, Azadirachta indica, and it has been shown to be the mainagent for combating mosquitoes (Su and Mulla, 1999; Suman et al.,2010). Azadirachtin is known to interfere in a wide range of physiolog-ical processes in mosquitoes and other insects. This botanical has been

omological Society and Malaysian Plant Protection Society. Published by Elsevier B.V.

176 A. Koodalingam et al. / Journal of Asia-Pacific Entomology 17 (2014) 175–181

reported to bind to chemoreceptors thereby resulting in deterrentand antifeeding responses (Mordue and Blackwell, 1993). Besides,azadirachtin modulates ecdysteroid and juvenile hormone titers, whichare well manifested by perceptible impairment of growth, moulting,ovarian development, oocyte ultrastructure, fecundity, and egg viability(Mordue and Blackwell, 1993; Su and Mulla, 1999; Lucantoni et al.,2006; Suman et al., 2010). Azadirachtin with such multitude or physio-logical effects has attracted greater attentionworldwide to explore its in-secticidal property against more than 200 insect species (Isman, 2008).In insects, esterases and phosphatases play important roles in variousphysiological processes (Krisch, 1971; Walter and Schütt, 1974; Urich,1994). Esterases are involved in digestion, neuronal conduction, repro-duction, metabolism of juvenile hormone, moulting and detoxificationof insecticides to less toxic metabolites (Urich, 1994; Wheelock et al.,2005). Phosphatases are essential for tissue growth, cellular differentia-tion, metabolism of carbohydrate, nucleotide and ATP generation(Walter and Schütt, 1974; Urich, 1994). Therefore, these two classes ofenzymes are considered as marker enzymes for assessment of impactof toxicants on biochemical or physiological processes of target insects(Smirle et al., 1996; Laranja et al., 2003; Nathan et al., 2008; Shekariet al., 2008; Smirle et al., 2010; Koodalingam et al., 2011, 2012; Pontualet al., 2012). Growing interest on application of neem-based productsgenerated large amount of scientific information on the effects of espe-cially azadirachtin on vector mosquitoes (Su and Mulla, 1998; Mullaand Su, 1999; Lucantoni et al., 2006; Ndione et al., 2006; Okumu et al.,2007; Lucantoni et al., 2010). However, systemic effects of neem-basedproducts with respect to modulations in esterases and phosphatases inlarvae and pupae of A. aegypti remain to be studied.

In mosquitoes, hemocytes are important components in cellular aswell as humoral immune responses by performing phagocytosis, encap-sulation, nodule formation and cytotoxicity (Hillyer, 2010). Hemocytesmount an effective immune challenge against invadingmicroorganismsand parasites through phagocytosis, thereby maintaining immunehomeostasis in mosquitoes (Hernández-Martinez et al., 2002; Blandinand Levashina, 2007). Among various hemocyte morphotypes detectedin systemic circulation of mosquitoes, mainly granulocytes rarelyplasmatocytes or oenocytoids have been reported to phagocytose vari-ous abiotic and biotic targets (Drif and Brehélin, 1983; Hernández-Martinez et al., 2002; Hillyer et al., 2003, 2005; Blandin and Levashina,2007). A few investigators have attempted to examine changes inhemocytic responses of the insects as a novel approach to elucidate theimpact of plant secondary metabolites on immuno-defence process. Inmost of such studies, alterations in hemogram, viability and ultrastruc-ture of hemocytes were assessed (Ayyangar and Rao, 1990; Azambujaand Garcia, 1992; Tikku et al., 1992; Peter and Anandakrishnan, 1995;Ayaad et al., 2001; Sharma et al., 2003, 2008). On the other hand, onlythree recent studies have examined the effect of botanicals on the func-tional capability of insect hemocytes by directly assessing the hemocyticability to bind and phagocytose foreign cell in vitro (Figueiredo et al.,2006; Zibaee and Bandani, 2010; Koodalingam et al., 2013). It is perti-nent to note that infectious fungal spores (microsporida), bacteria(Wolbachia) and virus (densovirus) possess inherent potency to knockdown mosquito population (Kurtz et al., 2000; Gu et al., 2011; Hurket al., 2012). The deleterious effect of these pathogens on mosquitoeshas led investigators to question whether they impair the immune sys-tem of mosquitoes, thereby making them more susceptible to infectionby opportunistic pathogens (James and Xu, 2012). Earlier studies havedemonstrated that the NeemAzal (a commercial azadirachtin formula-tion) exhibits anti-mosquito activity by reducing the survival, fecundityand adult emergence of three important vector mosquito species(Boschitz and Grunewald, 1994; Gunasekaran et al., 2009). However,its impact on biochemical marker enzymes and hemocyte-mediated cel-lular immune responses of the vector mosquitoes remains to be investi-gated. Therefore, the objective of the current study was focused on acomparative analysis of the effect of NeemAzal on two marker enzymes(esterases and phosphatases) as well as viability and phagocytic activity

of hemocytes of larvae and pupae of dengue vector mosquito Aedesaegypti.

Materials and methods

Fine chemicals and reagents

Acetylthiocholine iodide, DTNB reagent (5-5-dithiobis 2-nitro benzoicacid), Fast blue salt, α- or β-naphthyl acetate were obtained fromHimedia (Mumbai, India), p-nitrophenyl phosphate, p-nitrophenoland α- or β-naphthol were purchased from Ranbaxy (Mohali, India).All other chemicals and reagents used were of the highest analyticalgrade.

Mosquito rearing

The eggs of A. aegypti obtained from the laboratory reared mosqui-toes were allowed to hatch out under the controlled laboratory condi-tions (Koodalingam et al., 2009). The newly hatched fourth instarlarvae and pupae were used in all the experimental studies. Mosquitolarvae were reared in plastic bowls filled with unchlorinated tapwater. Larval foodwas supplied daily containing amixture of powdereddog biscuit and brewer's yeast at 3:1 ratio.

Preparation of NeemAzal stock solution

The botanical biocidal formulation NeemAzal T/S 1.5 EC waspurchased from E.I.D. Parry, Chennai, India. This neem formulationcontains 10,000 ppm of azadirachtin. Based on initial stock solution(10,000 ppm) various test concentrations were freshly prepared bysuitably diluting the NeemAzal with unchlorinated tap water.

Toxicity assay

The test media were prepared with different concentrations ofNeemAzal: 1, 2, 5, 10, 15 and 20 ppm, for the exposure of the larvaeand pupae. Batches of 10 newly hatched fourth instar larvae or pupaewere introduced into 50 ml of test medium. This experiment was per-formed with five replicates. Control containers received equal volumeof tap water instead of NeemAzal. The percentage of mortality of larvaeand pupae was recorded after 24 hour exposure period.

Preparation of whole body homogenates

The live control fourth instar larvae and pupae as well as theNeemAzal exposed larvae and pupaewerewashedwith double distilledwater, and the adhering water was completely removed from the bodysurface by blotting with tissue paper. The larvae and pupae (each 10individual) were separately homogenized in eppendorf tubes using aTeflon hand homogenizer in 500 μl of ice-cold 0.9% saline for eventualestimation of total proteins, esterases and phosphatases activity. Forqualitative analysis of esterase and phosphatases by native PAGE,the whole body homogenates of control and exposed larvae as wellas pupae were prepared as described earlier, except that 100 μl ofTris-glycine (pH 8.3) was used as homogenization buffer.

Quantitative analysis of biochemical constituents

Determination of protein concentration. The proteins present in thehomogenates of larvae and pupae were first precipitated with 80% eth-anol and the proteins present in the precipitate were estimated by themethod of Lowry et al. (1951) using bovine serum albumin as standard.

Acetylcholinesterase assay. The acetylcholinesterase activity wasdetermined by the procedure of Ellman et al. (1961) as described inKoodalingam et al. (2012) using acetylthiocholine iodide as a substrate.

Fig. 1. Toxicity of various concentrations of NeemAzal on fourth instar larvae and pupae ofthe vector mosquito Aedes aegypti.

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Briefly, 50 μl of larval or pupal homogenate was dissolved in 450 μl ofsodium phosphate buffer (100 mM; pH 7.5). To each reaction mixture50 μl of DTNB (10 mM) and 50μl of acetylthiocholine iodide(12.5 mM) were added and incubated at room temperature for 5 min.The optical density of the sample was read at 400 nm using Cecil CE7200 spectrophotometer against suitable blank.

Carboxylesterase assay. The activity of α and β carboxylesterase in thelarvae and pupae was measured by the method of Van Asperen(1962) with suitable modifications. Briefly, 100 μl of homogenateswere incubated with 900 μl of sodium phosphate buffer (100 mM,pH 7.0) containing 250 mM of α-naphthyl and β-naphthyl acetate re-spectively, for 30 min at room temperature. To each reaction mixture,400 μl of 0.3% fast blue B in 3.3% SDS were added to stop the enzymaticreaction and the colourwas allowed to develop for 15 min at room tem-perature. The optical density of the sample was read at 588 nm againstthe respective reagent blank in Cecil CE 7200 spectrophotometer.

Acid and alkaline phosphatase assays. The levels of acid and alkalinephosphatases in the larval and pupal homogenates were measured bythemethod of Asakura (1978), with slightmodifications. The acid phos-phatase activity was estimated by mixing 100 μl of larval or pupalhomogenate with 400 μl of 50 mM sodium acetate-acetic acid bufferat the optimal pH of 4.0. For the estimation of alkaline phosphatase ac-tivity, 100 μl of homogenate was mixed with 400 μl of 50 mM Tris–HClbuffer at the optimal pH of 8.0 and mixed with 500 μl of 15 mMp-nitrophenyl phosphate. After incubation for 15 min at 37 °C inwater bath, the enzymatic reaction was stopped by adding 100 μl of0.5 NNaOH solution and centrifuged (4000 ×g; 5 min). The absorbanceof the resulting clear supernatant was read at 440 nm against suitablereagent blank.

Qualitative analysis of biochemical constituentsThe profiles of ß-carboxylesterase, acid and alkaline phosphatases in

the whole body homogenates of fourth instar larvae and pupae wereanalysed in discontinuous polyacrylamide gel electrophoresis (PAGE)under non-denaturing conditions. This was performed using 3%stacking gel (pH 6.7) and 7% separating gel (pH 8.9) in Tris-glycinebuffer (pH 8.3). Samples of the whole body homogenates (each 50 μl)of control and experimental larvae or pupae were electrophoresed at aconstant current of 4 mA per sample and at 10 °C on a slab gel. Afterelectrophoresis, the gels were appropriately stained for detection ofß-carboxylesterase or acid and alkaline phosphatase activity.

Detection of ß-carboxylesterase activity. The electrophoretically separatedprotein bands with β-carboxylesterase activity were detected in the gelfollowing the method of Argentine and James (1995). Accordingly, thegel was first incubated with phosphate buffer (20 mM, pH 7.0) for15 min at RT. After decanting the buffer, the gel was then re-incubatedfor 30 min at RT with freshly prepared β-naphthyl acetate + Fast blueB solution for detection of β-carboxylesterase. The gels were extensivelywashed with distilled water and stored in 7% acetic acid.

Detection of acid and alkaline phosphatase activity. The electrophoreticallyseparated protein bands with acid or alkaline phosphatase activity weredetected in the gels following the method of Houk and Hardy (1984).Accordingly, the gel was first incubated with acetate buffer at pH 4.6(for larval homogenate) or 4.0 (for pupal homogenate) for 15 min atRT for detection of protein bands with acid phosphatase activity.After decanting the buffer, the gel was immersed in freshly preparedα-naphthyl phosphate + Fast blue B solution in 50 mM acetate bufferat respective pH and incubated for 2 h at 37 ºC on awater bath. The alka-line phosphatase activitywas detected after incubating the gel in Tris–HClbuffer (50 mM, pH 8.0) for 15 min at RT. After decanting the buffer, thegel was immersed in α-naphthyl phosphate + Fast blue B solutionfreshly prepared in the same buffer and then re-incubated for 2 h at

37 ºC on a water bath. The gels were extensively washed with distilledwater and stored in 7% acetic acid.

Hemocyte collection. Thehemocyteswere collected from live control andNeemAzal treated fourth instar larvae and pupae of A. aegypti as well aspreparation of hemocyte monolayer was performed according to themethod described by Koodalingam et al. (2013).

Cell viability assay. The viability of hemocytes recovered from controland exposed larvae and pupae was assessed by exclusion of trypanblue dye under Axioskop 2 plus microscope (Carl Zeiss, Germany). Thehemocyte monolayer was incubated for 1 h in the moist chamber. Thedead hemocytes were visualized by adding 100 μl of 0.1% trypan bluesolution in 10 mM phosphate buffered saline (pH 7.2; 300 mOsm)and incubated for 5 min later were washed with PBS. The live anddead cellswere counted in 10 randommicroscopic filed under Axioskop2 plus microscope.

Phagocytosis assay. Thephagocytic activity of hemocytes recovered fromcontrol and experimental larvae and pupae was measured using yeasttarget in vitro as described in Koodalingam et al. (2013). Briefly, 50 μlof heat killed target yeast cells (1.2 × 108 cells/ml) were overlaid onhemocyte monolayer and incubated in moist chamber for 30 min at23 ºC. The non-injested yeast cells were removed by gentle washingwith PBS andfixedwith 2% formaldehyde solution for 5 min. Phagocyticactivity against target yeast was measured by inspecting at least 100hemocytes under phase optics of Axioskop 2 plusmicroscope. The larvalor pupal hemocyte that was ingested, at least one yeast was counted asa positive one. The percentage of phagocytosis was calculated bycounting the number of positive hemocytes versus total number ofhemocytes × 100.

Statistical analysis. Statistical analysis of the experimental data wascarried out by student's t-test (SPSS version 9.0 software package).The acceptance level of statistical significance was p b 0.05 in allinstances and the results are expressed asmean ± SD of five replicates.

Results

In order to assess the impact of NeemAzal on marker enzymes andhemocytes of larvae and pupae of A. aegypti, the toxicity assay wasfirst performed. As depicted in Fig. 1, NeemAzal exposure inflicted100% mortality of both fourth instar larvae and pupae of A. aegyptiat 20 ppm within 24 h. The LC50 values of 6.82 ppm and 4.77 ppmwere recorded, respectively, for these two developmental stages of themosquito.

The effect of NeemAzal on total protein level in whole bodyhomogenates of larvae and pupae was examined by exposing the test

Fig. 2. The response of ß-carboxylesterase isoforms in the whole body homogenates ofcontrol (C) andNeemAzal exposed (E) larvae and pupae of A. aegypti. 50 μl of homogenatewere separated on a 7% polyacrylamide gel, and the esterase activity was stained with ß-naphthyl acetate + Fast blue B solution. Arrows indicate isoforms that are showing per-ceptible differences under exposure.

178 A. Koodalingam et al. / Journal of Asia-Pacific Entomology 17 (2014) 175–181

organisms to minimal lethal concentration (2 ppm) for 24 h (Table 1).NeemAzal exposure significantly altered the total protein level oflarvae which increased (30%; p b 0.01) from control value of 1.61 mgprotein/ml to 2.1 mg protein/ml in treated larvae. The protein level inNeemAzal-exposed pupae did not significantly change (p N 0.05).

The specific activities of esterases and phosphatases of control andexperimental larvae and pupae of A. aegypti are presented in Table 1.Activity of acetylcholinesterase was found to be remained almostsame in control and NeemAzal-exposed larvae (p N 0.05). However,the exposure of pupae to NeemAzal drastically increased (116%;p b 0.01) acetylcholinesterase activity. The level of α-carboxylesteraseactivity was significantly elevated in NeemAzal-treated larvae (121%;p b 0.001), whereas pupal enzyme activity was decreased (34%;p b 0.05). The exposure of larvae and pupae to NeemAzal resulted insignificant increase in the level of β-carboxylesterase activity 46%(p b 0.01) and 12% (p b 0.05), respectively. The NeemAzal exposuresignificantly increased the activity of acid phosphatases in larvae(62%; p b 0.01) as well as pupae (43%; p b 0.01). Similarly, exposureof larvae, but not pupae (p N 0.05), to this biocide significantlyincreased (37%; p b 0.01) the alkaline phosphatase activity over theirrespective controls.

The qualitative analysis of β-carboxylesterase activity revealed thatNeemAzal exposure significantly modulated the larval isoenzymic pro-file by down regulating twoprotein bands and up regulating at least twoprotein bandswith β-carboxylesterase activity, but no suchmodulationwas noticed in pupae (Fig. 2). As shown in Fig. 3, the intensity of proteinbandswith acid and alkalinephosphatase activitywas slightly increasedin NeemAzal-treated larvae. By contrast, these two enzymes did notshow any response in the toxicant exposed pupae.

Trypan blue dye exclusion test showed that more than 97% ofhemocytes from control larvae and pupae were viable under in vitrotest conditions (Fig. 4). However, the viability of hemocytes of experi-mental larvae and pupae of A. aegypti showed a significant reduction.The hemocytes from both larvae and pupae were susceptible toNeemAzal exposure as revealed by 18 and 16% reduction in cell viability(p b 0.01), respectively. In phagocytosis assays, an average of 16%hemocytes from control larvae contained at least one injested yeastcell whereas only 8% hemocytes expressed phagocytic activity inNeemAzal-exposed larvae (Fig. 5; p b 0.01). The phagocytic responseof hemocyteswas significantly decreased from16% to 9% upon exposureof pupae to NeemAzal (p b 0.01).

Discussion

The present study focuses the impact of NeemAzal (a commercialazadirachtin formulation) onbiochemical and immunological parametersof fourth instar larvae and pupae of the vector mosquito, A. aegypti.The NeemAzal exposure inflicted 100% mortality of fourth instar larvaeand pupae of A. aegypti with LC50 values of 6.82 ppm and 4.77 ppm,

Table 1Effects of NeemAzal on total protein, esterases and phosphatases of larvae and pupae of A. aegypreparations. NS indicates not statistically significant difference between control and experime

Biochemical parameters tested Larvae

Control NeemAzal exposed

Total protein(mg protein/ml of homogenate)

1.610 ± 0.058 2.117 ± 0.108

Acetylcholinesterase(mM ACT released/min/mg protein)

0.031 ± 0.003 0.040 ± 0.005

α-Carboxylesterase(μM α-naphthol released/min/mg protein)

6.208 ± 0.744 13.75 ± 1.117

β-Carboxylesterase(μM β-naphthol released/min/mg protein)

100.89 ± 13.16 147.32 ± 20.86

Acid phosphatase(mM p-nitrophenol released/min/mg protein)

0.295 ± 0.032 0.479 ± 0.076

Alkaline phosphatase(mM p-nitrophenol released/min/mg protein)

1.11 ± 0.08 1.52 ± 0.177

respectively. These findings demonstrate the susceptibility of both larvaeand pupae to this neem-based formulation, in which the pupae are morevulnerable. Many earlier investigators reported that mosquito larvaewere highly susceptible than pupae to various botanical biocides(Lapcharoen et al., 2005; Chowdhury et al., 2008; Koodalingam et al.,2009). On the other hand, methanolic extract of Atlantia monophyllaexerted higher mortality of pupae than the fourth instar larvae ofA. aegypti (Sivagnaname and Kalyanasundaram, 2004). Commercialneem formulations such as Neemarin (Vatandoost and Vaziri, 2004)and NeemAzal (Gunasekaran et al., 2009) were found to inflict higherpupicidal activity during cumulative exposures initiatedwith the third in-star larvae of Anopheles stephensi and A. Aegypti, respectively. However,the present study clearly demonstrated that even short-term exposure(24 h) to NeemAzal efficiently killed pupae of A. aegypti. The mosquitopupae, unlike the larvae, do not feed, and it is, therefore, plausible thatNeemAzal might have caused lethal effect through contact mode oftoxicity by binding to pupal exoskeletal structures.

Previous studies have shown that azadirachtin exposure significantlydecreased the total protein levels of various insect pests, namely, thepupae of Spodoptera litura, Ostrinia furnacalis, and fourth instar larvaeof Plodia interpunctella (Huang et al., 2004, 2007; Rharrabe et al., 2008).Two dimensional gel electrophoretic analysis revealed that azadirachtintreatment not only affected the expression of some proteins, but alsoup regulated certain proteins in the adults of Schistocerca gregaria(Annadurai and Rembold, 1993), pupae of S. litura and pupae of

pti. The value represents mean ± SD of five determinations using samples from differentnt (p N 0.05).

Statistical significance Pupae Statistical significance

Control NeemAzal exposed

p b 0.001 2.451 ± 0.336 2.250 ± 0.056 NS

NS 0.050 ± 0.007 0.109 ± 0.010 p b 0.01

p b 0.001 13.51 ± 1.220 8.886 ± 0.524 p b 0.05

p b 0.01 202.82 ± 11.44 176.64 ± 9.476 p b 0.05

p b 0.01 0.277 ± 0.044 0.396 ± 0.021 p b 0.01

p b 0.01 1.244 ± 0.119 1.054 ± 0.127 NS

Fig. 3. The response of acid and alkaline phosphatase isoforms in the whole body homog-enates of control (C) and NeemAzal exposed (E) larvae and pupae of A. aegypti. 50 μl ofhomogenate were separated on a 7% polyacrylamide gel, and the phosphatase activitywas stained with α-naphthyl phosphate + Fast blue B solution under two different pH(seeMaterials andmethods section). Arrows indicate isoforms that are showing percepti-ble differences under exposure.

Fig. 5. Impact of NeemAzal on in vitro phagocytic activity of hemocytes of fourth instar lar-vae and pupae of the vector mosquito Aedes aegypti. Each bar represents mean ± SD offive determinations using samples from different preparations. Asterisk indicates statisti-cally significant difference between control and experimental larvae andpupae (p b 0.01).

179A. Koodalingam et al. / Journal of Asia-Pacific Entomology 17 (2014) 175–181

O. furnacalis (Huang et al., 2004, 2007), thereby indicating modulatoryeffects of azadirachtin in protein metabolism. In our study, the shortterm (24 h) exposure of NeemAzal at 2 ppm significantly increased thetotal protein level of larvae, but not pupae. These observations suggestthat the chemical compound(s) present in the neem formulation testedmight effectively modulate the protein metabolism in the larvae ofA. aegypti.

A number of earlier studies have demonstrated qualitative and quan-titative changes of esterases in insect pests and vectors upon exposure tovarious botanicals (Smirle et al., 1996; Ortego et al., 1999;Wanchun et al.,1999; Breuer et al., 2003; Laranja et al., 2003; Nathan et al., 2008;Koodalingam et al., 2011; Pontual et al., 2012). The elevation in thelevel of esterases is considered as a common mechanism in insects to-wards development of resistance against many chemical insecticides(Polson et al., 2010). In the present study, differential responses in theactivity of esterases such as acetylcholinesterase, α-carboxylesterase,and β-carboxylesterase were recorded in fourth instar larvae and pupaeof A. aegypti following their exposure to NeemAzal. The low sensitivityof acetylcholinesterase to insecticides and subsequent increase in theactivity of this enzyme upon exposure of the target insects to such insec-ticides are well correlatedwith development of resistance inmany insectspecies (Fournier et al., 1992; Zhu andGao, 1999; Yang et al., 2008). How-ever, our investigation revealed that the activity of acetylcholinesterasewas significantly higher in NeemAzal-treated pupae whereas this en-zyme in larvae did not respond to the exposure. Despite an increase in

Fig. 4. Impact of NeemAzal on viability of hemocytes of fourth instar larvae and pupae ofthe vector mosquito Aedes aegypti. Each bar represents mean ± SD of five determinationsusing samples from different preparations. Asterisk indicates statistically significantdifference between control and experimental larvae and pupae (p b 0.01).

the level of acetylcholinesterase, the pupae of A. aegypti were found tobe more susceptible to NeemAzal exposure, and exact reasons for thisdiscrepancy are not clear.

Previous studies have shown that exposure of a wide range of targetinsects to various botanicals have decreased the level of esterases(Smirle et al., 1996; Ortego et al., 1999; Wanchun et al., 1999; Breueret al., 2003; Nathan et al., 2008; Koodalingam et al., 2011). The resultsof the present study also show varying pattern of alterations in theactivities of α-carboxylesterase, and β-carboxylesterase in larvae andpupae of A. aegypti upon exposure to NeemAzal. The NeemAzal treat-ment significantly elevated the levels of α- and β-carboxylesterases inthe larvae, but both these enzymes declined in the pupae. All thesefindings taken together clearly demonstrate the differential responsesofα- andβ-carboxylesterases in larvae and pupae of A. aegypti upon ex-posure to theneem formulation tested in this study. It is notable that theexposed larvae elevated the levels of two forms of carboxylesteraseswhich can be attributed to the physiological efforts of mosquito larvaeto detoxicity the toxic component(s) present in NeemAzal.

Phosphatases are considered another important biochemicalmarkerenzyme to assess the systemic effect of botanical biocides on targetinsects, because these enzymes are known to involve in diverse physio-logical functions. The exposure of insecticidal lectin from Galanthusnivalis and Canavalia ensiformis significantly increased the level of alka-line phosphatase activity in larvae of Lacanobia oleracea as comparedto larvae fed on diet without lectins (Fitches and Gatehouse, 1998). Bycontrast, exposure of azadirachtin to insect pests such as Cnaphalocrocismedinalis and S. litura, significantly lowered the levels of both acid andalkaline phosphatases (Nathan et al., 2005a,b). Recently, Koodalingamet al. (2011) demonstrated distinct variations in the levels of acid andalkaline phosphatases of larvae and pupae of A. aegypti exposed to ex-tract of soapnut Sapindus emarginatus. The short-term treatment of lar-vae of A. aegypti with NeemAzal significantly elevated the level of bothacid and alkaline phosphatases. In pupae, the exposure of NeemAzal en-hanced the level of only acid phosphatase activity. It is notable to recallthat the larvae of A. aegypti are less susceptible than pupae against expo-sure to NeemAzal. The results of quantitative analysis of phosphatasessuggest that the ability of the larvae, but not pupae, to up regulate thelevel of alkaline phosphatase might enable them to tolerate the toxicityof components in the neem formulation tested.

Few studies have qualitatively examined esterases and phosphatasesand demonstrated that insecticidal botanicals affect the isoenzyme pro-files of these enzymes of A. aegypti (Laranja et al., 2003; Koodalingamet al., 2011). Prolonged exposure to caffeine and coffee ground extractshas been reported to drastically affect the five isoenzymes esterases inthe larvalA. aegypti (Laranja et al., 2003).Moreover, short-termexposureto soapnut S. emarginatus aqueous extract at minimal lethal

180 A. Koodalingam et al. / Journal of Asia-Pacific Entomology 17 (2014) 175–181

concentration has modulated the isoenzymic profiles of esterases andphosphatases in the larvae and pupae of A. aegypti (Koodalingam et al.,2011). The NeemAzal with potent antimosquito activity significantly af-fected the isoenzymes of β-carboxylesterase, acid and alkaline phospha-tases in the larvae, and only acid phosphatase profile was altered in thepupae of A. aegypti. In general, the outcome of this qualitative analysisof various enzymes supports the quantitative data obtained from thelarvae and pupae exposed to NeemAzal. Our findings provide evidencethat the two distinct developmental stages of mosquito (larvae andpupae) employ different biochemical machinery to overcome the toxiceffects of the neem-based formulation tested in this investigation.

Hemocytes of vector mosquitoes are considered to be a potentialcandidate in innate immune responses by primarily participating inthe defence against parasites and pathogens (Hernández-Martinezet al., 2002; Hillyer et al., 2003; Blandin and Levashina, 2007). Phagocy-tosis is an evolutionary conserved innate cellular defence reactionagainst invading parasites and microbial pathogens (Lowenberger,2001; Blandin and Levashina, 2007). Any changes in hemocytic functiondirectly affect the physiologic and immunological status of the mosqui-to. Hemocyte-mediated cellular immune functions are valuable andsensitive indicators for assessment of toxicity of insecticides on insectpests and vectors. A few previous studies have reported the impact offew botanical biocides on the immune functions of insect hemocytes.Rhizomeoil fromAcorus calamushas been shown to affect the hemocyteviability and hemogram of tobacco armyworm S. litura (Sharma et al.,2008). The exposure of fifth instar nymphs of Rhodnius prolixus toazadirachtin affected the hemocytic responses by decreasing cellviability as well as phagocytosis (Figueiredo et al., 2006). Similarly,Zibaee and Bandani (2010) reported that the extract from Artemisiaannua reduced the phagocytic activity of hemocytes of the insectpest Eurygaster integriceps. Recently, Koodalingam et al. (2013) alsoobserved reduction in phagocytic response of hemocytes from fourthinstar larvae and pupae ofA. aegypti uponexposure to extract of soapnutS. emarginatus. The present study indicated that theNeemAzal exposuresignificantly affected viability and phagocytic ability of hemocytes fromboth the larvae and pupae of A. aegypti. These findings demonstrate thatthe exposure to NeemAzal inflicts an immuno-suppressive state inlarvae and pupae of vector mosquito A. aegypti.

Based on all the findings made in this study, we conclude that theneem-based insecticide NeemAzal exerted high mortality of pupae,modulated the levels of various detoxification enzymes and suppressedthe immune function of hemocytes from fourth instar larvae and pupaeof A. aegypti. The lethal effect of NeemAzal on two distinct developmen-tal stages of mosquito alongwith changes in important biochemical en-zymes and inflicting immuno-suppressive state by reducing phagocyticresponse of hemocytes suggest that this neem-based formulation can beeffectively used in integrated vector control programmes especially toovercome the usual problem of resistance development bymosquitoes.

Acknowledgments

We are grateful to Dr. K. Venkatesan, Principal, and Dr. B. Ramesh,Head, Department of Biotechnology, SSASC, Kanchipuram, for providingthe facilities and encouragement. We thank Professor P. Mullainadhan,Department of Zoology, University of Madras for use of his microscopyfacility.

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