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Use of Fluorescence, a Novel Technique to Determine Reductionin Bemisia tabaci (Hemiptera: Aleyrodidae) Nymph Feeding WhenExposed to Benevia and Other InsecticidesAuthor(s): Rachel Cameron , Edward B. Lang , I. Billy Annan , Hector E. Portillo ,and Juan M. AlvarezSource: Journal of Economic Entomology, 106(2):597-603. 2013.Published By: Entomological Society of AmericaURL: http://www.bioone.org/doi/full/10.1603/EC12370

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ARTHROPODS IN RELATION TO PLANT DISEASE

Use of Fluorescence, a Novel Technique to Determine Reduction inBemisia tabaci (Hemiptera: Aleyrodidae) Nymph Feeding When

Exposed to Benevia and Other Insecticides

RACHEL CAMERON, EDWARD B. LANG, I. BILLY ANNAN, HECTOR E. PORTILLO,AND JUAN M. ALVAREZ1

DuPont Crop Protection, StineÐHaskell Research Center, 1090 Elkton Road, Newark, DE 19711

J. Econ. Entomol. 106(2): 597Ð603 (2013); DOI: http://dx.doi.org/10.1603/EC12370

ABSTRACT The sweet potato whiteßy, Bemisia tabaci (Gennadius), is an economically importantpest in the United States and other countries. Growers in many places rely on the use of insecticidesto reduce populations of B. tabaci. However, insecticides may take a few days to cause B. tabacimortality and some do not reduce feeding before death. Earlier reduction of feeding of whiteßieswould decrease the physiological effects on plants, reduce the production of sooty mold and poten-tially reduce the transmission of viruses. Measuring the reduction in feeding after the exposure of B.tabaci to an insecticide has proven difÞcult. This series of laboratory experiments demonstrate theusefulness of ßuorescence in determining B. tabaci feeding cessation. Fluorescein sodium salt issystemically transported in the xylem from the roots to the plant leaves and absorbed by B. tabacinymphs feeding on these plants. Nymphs start ßuorescing shortly after the cotton plant root systemis submerged in the ßuorescein sodium salt. Using this novel technique, the effect of three insecticideswith different modes of action, cyantraniliprole, imidacloprid, and spirotetramat on B. tabaci wasevaluated and compared to determine reduction in feeding. Results indicate that B. tabaci nymphsfeeding on a plant treated with Benevia have a signiÞcant reduction of feeding when compared withnymphs feeding on plants treated with imidacloprid or spirotetramat. Both Benevia and spirotetramatcaused signiÞcant nymphal mortality by 48 h after exposure. This novel technique will be useful todemonstrate the feeding cessation or reduction in feeding produced by different insecticides in severalsucking insect groups.

KEY WORDS ßuorescein sodium salt, Cyazypyr, cyantraniliprole, imidacloprid, spirotetramat

The sweet potato whiteßy, Bemisia tabaci (Genna-dius), is perhaps the most economically importantwhiteßy pest in the United States and other countries,affecting multiple agricultural crop groups includingfruiting vegetables, Brassica spp. and cucurbits (Jones2003). This whiteßy is adapted to a wide range oflatitudes and climates and affects crops with its feed-ing in three ways: 1) Direct damage is caused byfeeding in the phloem of leaves and subsequentlyreducing the plant vigor and yields. 2) When adultsand nymphs of B. tabaci feed on plants, they excretehoneydew. Honeydew provides a substrate for thegrowth of sooty mold fungi, which affects the qualityof produce and reduces yields, and 3) B. tabaci isknown to transmit 111 plant viruses including severalspecies of begomoviruses (Jones 2003).

Additionally, several disorders have been linked tofeeding by whiteßies. Feeding by immature B. tabacihas been linked to silverleaf disorder in squash (Schus-ter et al. 1991). Even squash varieties that are resistantto the silverleaf disorder show decreased chlorophyll

levels, plant height, fruit quality and yield when ex-posed to whiteßies (McAuslane et al. 2004, Chen et al.,2004). A close correlation between whiteßy nymphand pupa counts and the presence of irregular ripen-ing disorder in tomatoes has also been observed(Schuster 2001). The irregular ripening disorder canoccur when plants are infested with whiteßies at thetomato maturing stage (McKenzie and Albano 2009),suggesting that control of whiteßy populations is cru-cial even when plants are near harvest.

As mentioned before, in addition to the direct feed-ing effects on the plant host, the presence of honey-dew can cause sooty mold to develop or interfere withthe processing of some crops. This is especially im-portant in crops such as cotton, where the whiteßyhoneydew produces a condition called “sticky cotton,”which can contaminate processing equipment andcause the yarn to break (Hequet and Abidi 2002).

Reducing feeding of whiteßies would decrease thephysiological effects on plants, reduce the productionof sooty mold and potentially reduce the transmissionof viruses. Research onB. tabacimanagement includesstudies on host plant resistance, biological control,1 Corresponding author, e-mail: juan.m.alvarez@usa.dupont.com.

0022-0493/13/0597Ð0603$04.00/0 � 2013 Entomological Society of America

cultural control, and chemical control. However,growers in the United States and other countries relyon insecticides to reduce populations of B. tabaci.Insecticides may take a few days to cause B. tabacimortality and some do not reduce feeding beforedeath. Measuring the reduction in feeding after theexposure of B. tabaci to an insecticide has provendifÞcult. To evaluate the reduction in feeding ofwhiteßies with the use of the above mentioned con-trols, researchers have used different methodologiesincluding the quantiÞcation of honeydew productionusing water sensitive paper (Blua and Toscano 1994,Yee et al. 1998), visualizing honeydew droplets withbromcresol green (He et al. 2011), or visualizing hon-eydew droplets on polyethylene (Henneberry et al.1999). Some studies also measured the amount ofsugars produced (Yee et al. 1998, Henneberry et al.1999). In this article, a novel evaluation tool to deter-mine the effect of insecticides on the feeding cessationof B. tabaci nymphs is presented. The objective of thisstudy was to determine whether B. tabaci insectswould ßuoresce when exposed to a ßuorescent dyeand also whether this ßuorescence could be used toindirectly assess the feeding status of B. tabaci afterbeing exposed to insecticide applications.

Materials and Methods

Insects. All B. tabaci nymphs used in the laboratorybioassays were reared at the DuPont StineÐHaskellResearch Center (Newark, DE) and maintained oncotton, Gossypium hirsutum L., in a growth chamberwithcontrolledenvironmental conditions(26.5 �1�C,70% relative humidity [RH] with a photoperiod of 16:8[L:D] h). Cotton plants with a single leaf were in-fested by placing them in a cage with whiteßy adults.Plants were periodically checked for eggs and oncethere were at least 30 eggs on the leaf of each plant,the adults were removed using a stream of air. Infestedplants were held in a growth chamber (28 [day]through 24 [night] �1�C, 60% RH, with a photoperiodof 16:8 [L:D] h) until insects reached the second orthird instar.Fluorescent Dye Exposure. Several dyes (ßuores-

cent and nonßuorescent) were evaluated to deter-mine if there was a suitable dye for plant and whiteßynymph uptake (data not shown). Fluorescein sodiumsalt solution (Fluka Analytical, St. Louis, MO) wasfound to be a suitable ßuorescent dye for plant andnymph uptake. Several concentrations were initiallytested todetermineanacceptableconcentration(datanot shown). The roots of cotton plants with thirdinstar nymphs were rinsed to remove the potting me-dia. Roots of these plants were placed in a 50 mlcentrifuge tube with 30 ml of 1,000 ppm ßuoresceinsodium salt solution or 30 ml of distilled water for thecontrol. The presence of ßuorescence in the leaf tis-sues observed under ultraviolet (UV) light producedby a Nikon Intensilight C-HGFI illuminator using aNikon BV-2A Þlter cube attached to a Nikon stereo-microscope model SMZ 1500 (Nikon, New York, NY),would indicate the plant had absorbed ßuorescein.

The presence of ßuorescence in the digestive systemof whiteßy nymphs that were present on the treatedplants would indicate that the insects had acquired thedye through feeding. Plants and nymphs were evalu-ated after 2 h for ßuorescence to determine if theywere ßuorescing using the technique described be-low. There were four plants per treatment.Evaluation Method to Determine Nymph Fluores-cence. The number of nymphs on a 0.62 cm2 section,(using a rectangular template) of leaf selected at ran-dom was determined under white light using the ste-reo microscope described above. Only sections of leafwith Þve or more nymphs were evaluated. The samesections of leaf were then observed under UV light todetermine if the nymphs were ßuorescing. Two sec-tions per leaf were evaluated under 25� magniÞcationas described above.Speed of Uptake and Concentration of FluoresceinSodium Salt. A preliminary experiment was con-ducted to determine how quickly the insects startedßuorescing after placing the plant in the ßuoresceinsodium salt solution. In the Þrst experiment, roots ofplants with second instar nymphs were placed in ei-ther 500 or 1,000 ppm of ßuorescein sodium salt so-lution as described above. Insects were evaluated forßuorescence after 60, 120, and 180 min. Five plants pertime point per concentration and two leaf sections perplant were used. Once it was determined that 1,000ppm of the ßuorescein sodium salt solution was thebest concentration to show the ßuorescence on thedigestive system of B. tabaci nymphs, a second exper-iment with this concentration was conducted to de-termine the speed of uptake of the ßuorescein sodiumsalt solution. Insects were evaluated for ßuorescenceat 15, 30, 60, and 120 min after exposure. Four plantsper time point and two leaf sections per plant wereused in this second experiment.Evaluation of Feeding, Cessation after the Applica-tion of Insecticides. The adaxial surface (upper leafsurface) of cotton plants in the abaxial surface, withsecond instar nymphs were sprayed with insecticidesusing a moving belt sprayer, TwinJet 8002E, 30 psi, 141feet/min (DuPont, Newark, DE). Three insecticideswith different modes of action were evaluated andcompared in this experiment: 1) Benevia (10 OD Cya-zypyr, cyantraniliprole, DuPont Crop Protection, Wil-mington, DE), a novel cross-spectrum second gener-ation anthranilic diamide insecticide that wasdiscovered by the DuPont Company, and has provento be effective at controlling economic sucking andchewing insect pests in many crops. 2) Provado 2 F(imidacloprid, Bayer CropScience, Research TrianglePark, NC) a nicotinic acetylcholine receptor agonist,and 3) Movento (spirotetramat, Bayer CropScience)an acetyl CoA carboxylase inhibitor (IRAC 2012). Thethree insecticides were applied at 600, 300, and 374ppm (active ingredient [AI]), respectively. Theseconcentrations were equivalent to the label rates:0.135 kg (AI) per hectare (0.12 lbs. [AI] per acre) forcyantraniliprole (proposed label rate; product not reg-istered in the United States yet), 0.071 kg (AI) perhectare (0.063 lbs. [AI] per acre) for imidacloprid

598 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 2

(Bayer CropScience 2010) and 0.088 kg (AI) per hect-are (0.078 lb [AI] per acre) for spirotetramat (BayerCropScience 2011) at 234 L/ha (25 gallons of waterper acre). All solutions were prepared the day of theexperiment in distilled water. No adjuvants or solventswere used in preparation of the solutions. Plants wereplaced in the ßuorescein sodium salt solution at fourtime points: 10 min, 4, 24, and 48 h after the spray.Plants were placed in ßuorescein immediately afterthe spray (10 min) to verify the insecticides did nothave an immediate effect. The next time point (4 h)was chosen to allow the insecticide enough time tomove into the leaf tissue. There were four plants pertime point per insecticide. Live nymphs in two leafsections per plant were evaluated for ßuorescence 120min after placing the plant in ßuorescein sodium saltsolution. At the 24 and 48 h time points the entire leaffor each treated and untreated plant was evaluated formortality. Nymphal mortality was recognized by themargin curling of the nymphs.Statistical Analyses.All percentage data were trans-

formed using the arcsine of the square root and ana-lyzed using a one way analysis of variance (ANOVA)(Proc � GLM). Statistical analyses and analyses forinteractions were performed in SAS (SAS Institute

2002Ð2008). Treatment means were separated usingTukeyÕs Studentized Range Test (� � 0.05).

Results

Fluorescent Dye Exposure. None of the plants orinsects in the water control showed ßuorescence. Theleaf tissue in the ßuorescein sodium salt treatmentshowed ßuorescence (Fig. 1). Additionally, all B.tabaci nymphs in the plants exposed to ßuoresceinsodium salt presented ßuorescence in their digestivesystem, indicating they were actively feeding (Fig. 2).Speed of Uptake and Concentration of FluoresceinSodium Salt. The ßuorescence was visible on the di-gestive systemofB. tabacinymphs fasterwith the1,000ppm concentration of ßuorescein sodium salt thanwith the 500 ppm concentration (Table 1). After 60min of exposure to the ßuorescein sodium salt 100%nymphswereßuorescing inthe1,000ppmconcentration(F � 21; P � 0.0019) versus 37% in the 500 ppm con-centration. Once the 1,000 ppm concentration wasdeemedthebestconcentrationtoshowtheßuorescenceon the nymphs, a second experiment conducted to de-termine the speed of uptake showed that a few nymphsstarted ßuorescing 15 min after exposure (Table 2).However, it took 30 min to observe 100% of the nymphs

Fig. 1. Cotton leaves with Bemisia tabaci whiteßynymphs. (A) Leaf with no ßuorescein sodium salt (left) andleaf with ßuorescein sodium salt (right) under white light.(B) Same leaves under UV light.

Fig. 2. Bemisia tabaci whiteßy nymphs under UV lightfeeding on a plant treated with the ßuorescein sodium saltunder 1.75� magniÞcation.

Table 1. Percentage of Bemisia tabaci nymphs fluorescingwhen feeding on cotton plants exposed to two concentrations offluorescein sodium salt

Fluorescein sodiumsalt concn

% ßuorescing � SE

60 min 120 min 180 min

500 ppma 37 � 15bc 100a 100a1,000 ppmb 100a 100a 98 � 2a

a n � 74, 63, and 93 for the 500 ppm at 60, 120, and 180 min,respectively.b n � 71, 60, and 91 for the 1,000 ppm at 60, 120, and 180 min,

respectively.c Averages followed by the same letter within a column are not

statistically different (TukeyÕs test P � 0.05).

April 2013 CAMERON ET AL.: USING FLUORESCENCE TO DETERMINE B. tabaci FEEDING 599

ßuorescing (F� 144; P� �0.0001). The progression ofdye in the plants was clearly visible with very little ßu-orescence showing after 15 min of exposure (Fig. 3).Evaluation of Feeding Cessation. The percentages

of B. tabaci nymphs ßuorescing after different expo-sure times to the insecticides tested are presented inTable 3. AllB. tabacinymphs on the insecticide treatedplants were ßuorescing at the beginning of the exper-iment (0 h), indicating they were actively feeding(F � 1; P � 0.43). However, observations at 4 h afterinsecticide exposure demonstrated a signiÞcant re-duction of nymphal feeding on the cyantraniliproletreated plants, when compared with the nymphs feed-ing on imidacloprid and siprotetramat treated plants(F � 21; P � �0.0001). The imidacloprid and spiro-tetramat treatments presented 94 and 100% of nymphsßuorescing, respectively (Table 3) versus 32% for cy-antraniliprole. The percentage of nymphs ßuorescingat 24 h after insecticide exposure was again signiÞ-cantly lower (26%) for the cyantraniliprole treatment,with 93 and 100% of nymphs feeding in the imidaclo-prid and siprotetramat treated plants, respectively(Table 3) (F � 24; P � �0.0001) (Fig. 4).

After 48 h of insecticide exposure there were notenough live nymphs to evaluate for feeding in thecyantraniliprole treatment. However, the imidaclo-prid and spirotetramat treated plants had 94 and 100%of nymphs feeding, respectively (F � 1; P � 0.41). Insummary, cyantraniliprole produced the fastest re-duction in B. tabaci nymph feeding. The absence ofßuorescence in the digestive system of nymphs thatwere on plants treated with cyantraniliprole indicatedthat the nymphs were not feeding. Neither spirote-

tramat nor imidacloprid showed a reduction in the B.tabaci nymph feeding. Most of the nymphs were ßu-orescing after 48 h of exposure to these insecticides,indicating that they were still feeding.

Cyantraniliprole had the highest mortality at 24 and48 h after treatment (Table 3). Nymph mortality 24 hafter exposure in the imidacloprid and siprotetramattreated plants was not signiÞcantly different to the onein the untreated control (F � 9.3; P � 0.0019). Theimidacloprid treatment presented the lowest mortal-ity 48 h after treatment (Table 3). Nymphal mortality48 h after treatment in the plants treated with spiro-tetramat was signiÞcantly higher than the one on theuntreated control (F � 244; P � �0.0001). However,all remaining live nymphs were still feeding (Table 3).

Discussion

Several techniques have been used to determine thefeeding behavior of whiteßy nymphs. One of thesetechniques is the Electrical Penetration Graph or EPG(Jiang and Walker 2003). EPG is a system that uses anelectric circuit that closes when insect species such asaphids, whiteßies, leafhoppers, or thrips insert theirstylets into the plant host. The circuit is visualized asa graph that shows different waveforms indicating adetailed account of feeding behaviors from salivationto ingestion and also where in the plant tissue theseevents are taking place (i.e., mesophyll, xylem, orphloem). Although the EPG provides very detailedinformation, not all the waveforms have been identi-Þed with the corresponding insect behavior (Jiang andWalker 2003), it is very labor intensive, and the num-ber of insects that can be evaluated is limited.

Most of the techniques currently available indi-rectly determine the reduction in feeding by quanti-fying the amount of honeydew produced by white-ßies. These techniques include collecting honeydewdroplets on polyethylene (Henneberry et al. 1999),water sensitive paper (Blua and Toscano 1994, Costaet al. 1999), and analysis of sugars collected (Nauen etal. 1998; Yee et al. 1998; Henneberry et al. 1999). Onestudy using water sensitive paper showed thatBemisianymphs did not start producing honeydew for 3Ð5 dafter hatching (Blua and Toscano 1994) while another

Table 3. Percentage of Bemisia tabaci nymphs fluorescing after 120 min of exposure to fluorescein sodium salt and percentagemortality after different times following exposure to insecticides

% ßuorescing � SE after different insecticide exposure timesa % mortalityb

0 h 4 h 24 h 48 h 24 h 48 h

Benevia 600 ppm 100 � 0ac 32 � 12b 26 � 11b *d 62 � 8a 97 � 1aImidacloprid 300 ppm 100 � 0a 94 � 4a 93 � 2a 94 � 6a 11 � 4b 4 � 1cSpirotetramat 374 ppm 100 � 0a 100 � 0a 100 � 0a 100 � 0a 36 � 12ab 81 � 7bControl 99 � 1a 95 � 3a 98 � 3a 100 � 0a 9 � 6b 0 � 0d

100% of the nymphs were ßuorescing after 30 min exposure to ßuorescein sodium salt (Table 2). However, 120 min exposure was used toaccount for a possible slower ingestion rate by the treated nymphs.a n for % ßuorescing 0 h � 51, 52, 55, 55; 4 h � 45, 47, 55, 52; 24 h � 50, 42, 49, 37; 48 h � *, 58, 54, 48 for Benevia, imidacloprid, spirotetramat,

and the control, respectively.b n for % mortality 24 h � 1054, 654, 718, 256; 48 h � 1123, 932, 908, 343 for Benevia, imidacloprid, spirotetramat, and the control, respectively.c Averages followed by the same letter within a column are not statistically different.d *, not enough live nymphs to evaluate ßuorescence.

Table 2. Percentage of nymphs fluorescing when feeding oncotton plants exposed to 1000 ppm of fluorescein sodium salt

No. of nymphs % ßuorescing � SE

15 min exposure 94 8 � 8ba

30 min exposure 133 100 � 0a60 min exposure 70 100 � 0a120 min exposure 69 100 � 0aControl 96 0 � 0b

a Averages followed by the same letter within a column are notstatistically different (TukeyÕs test P � 0.05).

600 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 2

study showed honeydew production began almost im-mediately (Costa et al. 1999). Honeydew dropletsproduced by second instar nymphs are very small (2.5mm2 � 10�3) (Costa et al. 1999) that may lead to thedifÞcultly of collecting the droplets on water sensitivepaper thereby producing dissimilar results even whenthe same insect species is studied by different researchlaboratories. Water sensitive paper can be a valuabletool fordetermining insect feedingbehaviorbut itmaynot be ideal when working with early instars thatproduce very small honeydew droplets. To the best ofour knowledge, the EPG or honeydew measurementare the only techniques currently used to determinethe effect of insecticides on the feeding of B. tabaci.

This study shows that ßuorescein sodium salt can beused to visualize the feeding of the B. tabaci nymphsand potentially other sucking insects too. To the bestof our knowledge this is the Þrst time ßuorescein

sodium salt has been used to visualize insect feeding.The 1,000 ppm ßuorescein sodium salt solution wasvisible in the majority of the nymphs at 30 min afterplacing the plant in the salt solution. However, eval-uation of feeding in the nymph feeding cessation assaydid not start until after 120 min, to account for apossible slower ingestion rate by the treated nymphs.Fluorescein sodium salt is systemically transported inthe xylem from the roots to the plant leaves and ab-sorbed by B. tabaci nymphs feeding on these plants. Itis possible that the dye is not restricted to the xylemand may also be present in the phloem. Using EPG, ithas been demonstrated that hemipteran insects suchas B. tabaci adults, Trialeurodes vaporariorum (West-wood) adults, andMacrosiphum euphorbiae (Thomas)adults spend the majority of the time feeding in thephloem; however, they also feed in the xylem (Jianget al. 1999, Lei et al. 2001, Pompon et al. 2011). It is

Fig. 3. Fluorescence of cotton leaves after exposure to ßuorescein sodium salt. AÐD are 15, 30, 60, and 120 min of exposure,respectively.

Fig. 4. Fluorescence of cotton leaves andBemisia tabacinymphs after exposure to ßuorescein sodium salt. (A) Is untreatedand (B) is 24 h after exposure to Benevia.

April 2013 CAMERON ET AL.: USING FLUORESCENCE TO DETERMINE B. tabaci FEEDING 601

uncertain how much time B. tabaci nymphs spendfeeding in the xylem. However, two of the insecticidestested, cyantraniliprole and imidacloprid, are consid-ered to be xylem mobile not phloem mobile. Becausecyantraniliprole showed a reduction in feeding as wellas whiteßy mortality it suggests that the nymphs spendat least part of the time feeding in the xylem.

This study demonstrated a reduction in percentageof nymphs feeding when the adaxial surface of cottonleaves was treated with cyantraniliprole. Contrary toprevious studies with imidacloprid that showed a re-duction in feeding with B. tabaci adults when exposedto leaf discs dipped in solution (Nauen et al. 1998, Heet al. 2011), this study did not show a signiÞcant re-duction in feeding with imidacloprid. This may bebecause of the fact that different life stages weretested (adults vs. nymphs) or to the different appli-cation method (leaf dip in the studies with adultsversus translaminar in these studies). Spirotetramatdid not show a reduction in feeding but did showmortality within 2 d, which is consistent with previousmortality data (Bruck et al. 2009). These data showthat insects that are affected by some insecticides maycontinue to feed until they die while other insecticidesmay prevent the insect from feeding before they die.

Cyantraniliprole, the active ingredient in Benevia,exhibits a novel mode of action, by selectively acti-vating the ryanodine receptors in insect muscles, re-sulting in rapid cessation of feeding in affected pestinsects (Sattelle et al. 2008). This causes reductions indirect feeding damage and also in the capability of theaffected pest insects to vector plant pathogens. Jacob-son and Kennedy (2011) recently demonstrated thatsoil applications of cyantraniliprole at 4.41 mg AI perplant to sweet pepper,Capsicum annuum L. seedlings,signiÞcantly reduced the transmission of Tomato spot-ted wilt virus (TSWV) by Frankliniella fusca (Hinds)when compared with the water-treated control. Theauthors mentioned thatbecausecyantraniliprolecauseda signiÞcant reduction in feeding injury, without a cor-responding increase in mortality of F. fusca adults at 5 dafter treatment, it is likely that antifeedant effects areresponsible for the decreased transmission of TSWV.Additional research on this subject is currently showingpositive results of using cyantraniliprole to reduce thetransmission of other viral and bacterial pathogens trans-mitted by whiteßies and other insects.

Acknowledgments

The authors would like to thank Joe Saienni and MaryKoechert for their technical assistance with these studies. Weare grateful to A. Lund and L. Teixeira, DuPont Crop Pro-tection, and other anonymous reviewers for their helpfulcomments on the manuscript.

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Received 6 September 2012; accepted 28 December 2012.

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