alpha mangostin_analisis larvicidal

8/11/2019 Alpha Mangostin_analisis Larvicidal

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VECTOR/PATHOGEN/HOSTINTERACTION, TRANSMISSION

The Biological Activity of-Mangostin, a Larvicidal Botanic MosquitoSterol Carrier Protein-2 Inhibitor

RYAN T. LARSON,

1

JEFFREY M. LORCH,

2

JULIA W. PRIDGEON,

3

JAMES J. BECNEL,

3

GARY G. CLARK,3 ANDQUE LAN1,4

J. Med. Entomol. 47(2): 249257 (2010); DOI: 10.1603/ME09160

ABSTRACT -Mangostin derived from mangosteen was identied as a mosquito sterol carrierprotein-2 inhibitor via high throughput insecticide screening.-Mangostin was tested for its larvicidalactivity against third instar larvae of six mosquito species, and the median lethal concentration valuesrange from 0.84 to 2.90 ppm. The residual larvicidal activity of -mangostin was examined undersemield conditions. The results indicated that -mangostin was photolytic with a half-life of 53 minin water under full sunlight exposure. The effect of-mangostin on activities of major detoxication

enzymes such as P450, glutathione S-transferase, and esterase was investigated. The results showedthat -mangostin signicantly elevated activities of P450 and glutathione S-transferase in larvae,whereas it suppressed esterase activity. Toxicity of-mangostin against young rats was studied, andthere was no detectable adverse effect at dosages as high as 80 mg/kg. This is the rst multifacetedstudy of the biological activity of-mangostinin mosquitoes. Theresults suggest that-mangostin maybe a lead compound for the development of a new organically based mosquito larvicide.

KEY WORDS sterol carrier protein-2, larvicide, mangostin, detoxication enzymes, mosquitoes

David Fairchild described mangosteen (Garciniamangostana L.) as the

queen of tropical fruit

in his

1930 book,Exploring for Plants(Fairchild 1930). Ac-cording to Fairchilds account, the juicy, sweet-tastingfruit drove Queen Victoriato offer100to anyonewhocould bring her one mangosteen. This feat would havebeen quite challenging in the 19th century, consider-ing G. mangostana is a tropical evergreenthat is mainlyfound in India, Myanmar, Sri Lanka, and Thailand(Jung et al. 2006). Almost a decade after Fairchildsaccount, the mangosteen received much scientic at-tention because of its xanthone-rich pericarp. Xan-thones are heterocyclic compounds and are biologi-cally active in numerous pathways (Fotie and Bohle

2006). Studies have found that xanthone derivativesfrom the pericarp of a mangosteen possess antiplas-modial (Mahabusarakam et al. 2006), anticancer (Na-kagawa et al. 2007), antimicrobial (Mahabusarakam etal. 1986), and antioxidant (Jung et al. 2006) properties.

-Mangostinis a mangosteen-derived xanthone thatwas identied during a high throughput insecticidescreen as a mosquito sterol carrier protein-2 (SCP-2)inhibitor (SCPI) (Kim et al. 2005). SCPIs are a novel

class of insecticides that target the SCP-2, which ispartially responsible for intracellular cholesteroltransport in insects (Larson et al. 2008, Lan and Mas-sey 2004, Blitzer et al. 2005, Kim et al. 2005, Dyer et al.2008). Cholesterol trafcking is essential for insectsbecause they are unable to synthesize cholesterol denovo, and, as a result, insects rely on dietary sourcesof cholesterol (Clark and Bloch 1959). Therefore, theinhibitionof cholesterol uptake andtransport has con-sequential effectson insects. It has been demonstratedthat SCPIs are toxic to mosquito and Manduca sextalarvae (Larson et al. 2008, Kim et al. 2005). Interest-ingly, SCP-2 is not critical for survival in vertebrates(Kannenberg et al. 1999, Fuchs et al. 2001). Moreover,-mangostin has very low mammalian toxicity (Sorn-prasit et al. 1987). The novel mode of action and lowmammalian toxicity combined with the fact that-mangostin is extracted from the nonedible pericarpof the mangosteen fruit suggest that it might be de-veloped as a promising organic insecticide.

Previous studies have found that -mangostin hasinsecticidal properties against dipteran, coleopteran,and hemipteran pests. Whereas Ee et al. (2006) iso-lated -mangostin and determined the 24-h medianlethal concentration (LC

50) to be 19.4 gml1 against

third instarAedes aegypti L., the mode of action of the

toxicological effects of-mangostin against this mos-quito species was not proposed. However, work per-formed in Thailand with the rice weevil (SitophilusoryzaeL.) and the brown plant hopper (NilaparvatalugensStal.) suggests that -mangostin inhibits ester-ase, acetylcholinesterase, and glutathione S-trans-

1 Department of Entomology, University of Wisconsin, Madison,WI 53706.

2 Molecular and Environmental Toxicology Center, University of

Wisconsin, Madison, WI 53706.3 U.S. Department of Agriculture-Agricultural Research Service,

Center for Medical and Veterinary Entomology, 1600S.W. 23rdDrive,Gainesville, FL 32608.

4 Corresponding author: Department of Entomology, University ofWisconsin, 1630 Linden Drive, Madison, WI 53706 (e-mail:[email protected]).

0022-2585/10/02490257$04.00/0 2010 Entomological Society of America


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ferase (GST) activities (Bullangpoti et al. 2004, 2006).In this study, we evaluated -mangostin as a SCPI formosquito control.-Mangostin was tested against sixspecies of mosquito larvae and one adult species todetermine the relative toxicity. The persistence ofbiological activity of-mangostin in sunlight was de-termined in a semield trial. We also revisited the

report that -mangostin possesses enzymatic inhibi-tory properties using a mosquito model.

Materials and Methods

Chemicals. All chemicals and reagents were pur-chased from Sigma-Aldrich (St. Louis, MO) andFisher Scientic (Pittsburgh, PA), unless otherwisespecied. All laboratory bioassays used a 50 mg ml1

stock solution of-mangostin (Gaia Chemical, Gay-lordsville, CT)in which-mangostin(98%purity)wasdissolved in dimethyl sulfoxide (DMSO). For the

semield trials, -mangostin (97% purity) was ob-tained from Indone (Hillsborough, NJ) and a 50 mgml1 stock solution was prepared by dissolving it inDMSO. Technical grade temephos was provided byClarke Mosquito Control (Schaumburg, IL). Teme-phos was dissolved in DMSO to prepare 1 mg ml1

stock solution.Mosquitoes. The Rockefeller strain of Ae. aegypti

was reared in an incubator in Russell Laboratorieslocated on theUniversityof Wisconsin (Madison, WI)campus. Hatching was induced by transferring a pieceof egg-covered lter paper to a 500-ml side arm asklled with250 mlofddH

20. The ask was placed under

a vacuum for 20 min before the hatched larvae weretransferred to a plastic tray containing 3000 ml ofddH20. The density was1000 larvae/3000 ml ddH20.The larvae were fed Tetramin sh food and reared inan incubator maintained at 26C, 70% RH, and with aphotoperiod of 16 h:8 h (L:D).

Anopheles stephensi, Anopheles gambiae,and Culexpipiens pipiens larvae were obtained from Dr. SusanPaskewitz (University of Wisconsin, Madison, WI).Mosquitoes were maintained in an incubator at 26C,7075% RH, and a photoperiod of14 h:10 h (L:D). Theeggs were then hatched in aluminum trays lled with

deionized water. The larvae were provided a diet ofVitaPro Plus sh food and brewers yeast at a 2:1 ratio,respectively.

Ae. aegypti (Orlando strain),Anophelesquadrimacu-latusSay, andCulex quinquefasciatusSay were rearedin the insectary of the Mosquito and FlyResearch Unitat Center for Medical and Veterinary Entomology(CMAVE), U.S. Department of Agriculture-Agricul-tural Research Service, according to the proceduresdescribed by Pridgeon et al.(2008).Ae. aegypti andAn.quadrimaculatushave been established in the insec-tary since 1952 from Orlando, FL strains.Cx. quinque-

fasciatus has been established in the insectary since1995 from a Gainesville, FL strain. Eggs were hatchedin a ask with deionized water, left overnight, andtransferred to a plastic tray containing distilled water.A powdered diet (2:1 pot belly pig chow:brewersyeast) was added to each tray. Mosquitoes were

reared in an environmental chamber at 2230C, 80%RH, and a photoperiod of 14 h:10 h (L:D).

Mammalian Toxicology Tests.Male Sprague Daw-ley rats, 20 21 d old (4045 g), were obtained fromHarlan (Indianapolis, IN) and housed individually inshoe box-type cages with wire lids. Rats were allowedfreeaccess to food (Product8604, TekladLaboratories

Rodent Diet, Madison, WI) and water throughout theexperiment, except when animals were fasted over-night before blood collection.

Animals were allowed to acclimate to their newenvironment for at least 3 d before initiation of theexperiment. The-mangostin solution for administra-tion was prepared by dissolving it in DMSO at a con-centration of 200 mg/ml. This was then diluted inDMSO to achieve the correct concentration for eachanimal. All animals, including controls, received solu-tions at a rate of 400 l/kg body mass during eachdosing.

Four different dosages were tested, as follows: 20mg/kg (n 4), 40 mg/kg (n 4), 60 mg/kg (n 4),and 80 mg/kg (n 4). The compound was adminis-tered orally on a daily basis for consecutive 5 d, fol-lowed by a period of 2 d without treatment. Thisregime was carried out for consecutive 26 d. Controlanimals received equivalent volumes by body mass ofthe solvent for-mangostin (DMSO;n 13) or ster-ilized distilled water (n 8). The water control wasimplemented to ensure that DMSO had no signicantimpact on the parameters being measured.

The physical condition of both treated and controlanimals was monitored daily. Animals were examinedfor signs of dehydration, lethargy, unresponsiveness,and stress (stress was noted by excessive porphyrindischarge from the eyes and nose). Animals were alsoweighed daily to check for signs of weight loss or tonote unusual patterns in growth rate.

Blood samples were collected 5 d after administra-tion of the compound began and every 7 d thereafter(i.e., days 5, 12,19,and 26). Animals were anesthetizedusing isourane, and the blood was collected from the

jugular vein. Blood was placed into heparinized tubes,and the plasma was extracted for analysis. The Uni-versity of Wisconsin School of Veterinary Medicine

(Madison, WI) analyzed the samples for serum levelsof alanine aminotransferase (ALT), a biomarker ofhepatocyte damage.

Seventy-Two-Hour Larvicidal Bioassays.Bioassaysfor the toxicity of -mangostin were carried outagainstsix species of mosquitolarvae. All ofthe specieswere tested at the third instar. Ae. aegypti (Rock-efeller), An. stephensi, and Cx. pipiens pipiens weretested in 60 ml of ddH

20 using 60 larvae per containeraccording to the methods described by Larson et al.(2008). The larvae used in these assays were main-tained in an incubator at 26C, 70% RH, and a photo-

period of 14 h:10 h (L:D).Ae. aegypti(Orlando),An.quadrimaculatus, and Cx. pipiens quinquefasciatuswere tested at CMAVE, based on a modied WorldHealth Organization method (WHO 2005). The bio-assays performed at CMAVE used 100 ml of deionizedwater and 20 third instar larvae per container, and

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were maintained at room temperature under constantlighting. In all assays, -mangostin was serially dilutedto six concentrations and fed a powdered diet everyother day. DMSO equivalents were used as the con-trol. The number of living and dead larvae wascounted 72 h after the start of assay.

Continuous Versus 24-h Exposure to -Mangostin

to Mosquito Larvae.The effect of continuous expo-sure to -mangostin versus 24-h exposure was in-vestigated at CMAVE using Ae. aegypti (Orlando).Biological assays were performed, as previously de-scribed, using 100mlofdeionized water, 20 third instarlarvae per container, and six concentrations of-man-gostin. However, in the 24-h treatment, larvae wereremoved from solutions of -mangostin after 24 h,washed twice in deionized water, and then placed in100 mlof deionized water for the remaining 48 h oftheexperiment. Controls consisting of equivalent concen-trations of DMSO were set up for both the 24-h ex-

posure treatment and continuous exposure treatment.Control larvae in the 24-h exposure treatment wereremoved from the DMSO solution and washed twicebefore being placed in 100 ml of deionized water. Thenumber of living and dead larvae was counted 72 hafter the start of the assay.

Mosquito Adult Bioassays. Adult bioassays wereperformed at CMAVE, as described by Pridgeon et al.(2008), with slight modications. -Mangostin wasdissolved in acetone to make a 100 mg ml1 stocksolution before being serially diluted to six concen-trations (3.13100 mg ml1). These concentrationswere selected after an initial screen determined that-mangostin hadbiologicalactivity in this range. Adult

Ae. aegypti(Orlando) were used for the assays andwere removed from a holding cage with an Insect Vac(BioQuip Products, Rancho Dominguez, CA) andplaced on a chill table so the females could be sepa-rated from the males. Thirty female Ae. aegyptiwereplaced in plastic cups covered with mesh screens for2448 h before the start of the bioassay. A cotton ballsoaked in a 10% sucrose solution was placed on top ofthe mesh screen. The 5- to 7-d-old adult mosquitoeswere anesthetized with CO

2for 30 s before being

transferred to a chill table (BioQuip Products, Rancho

Dominquez, CA). A 700 series syringe and PB 600repeating dispenser (Hamilton, Reno, NV) were usedto apply 0.5l of-mangostin solution topically to thedorsal thorax of the females. An equivalent amount ofacetone was applied as a control. After the topicalapplication, the mosquitoes were immediately trans-ferred back into the plastic cups with a mesh top andprovided with a cotton ball soaked in a 10% sucrosesolution. Mortality was recorded 48 h after treatment.

Persistence of Biological Activity of-Mangostin inShade and Laboratory Conditions. The methods ofPerez et al.(2007) were slightly modied to determine

the persistence of-mangostin in the shade and lab-oratory conditions. This experiment was performed atCMAVE from 28 July 2008 to 7 August 2008. A 40 gml1 solution of-mangostinwas prepared in 5-gallonbuckets lined with high-density molded polyethyleneinserts (Associated Bag, Milwaukee, WI) with a total

volume of 5 L of deionized water. Containers in theshade treatment were carried outside between 9:00and 10:00 a.m., covered with a plastic roof and anultraviolet (UV)-resistant tarp, whereas the contain-ers in the laboratory were kept in a room with nowindows and maintained at 22C. An initial sample(time 0) was taken before the buckets were carried to

respective treatment locations. Each treatment in-cluded four containers that were independently sam-pled at time 0 (initial), 6 h, 12 h, 48 h, 120 h, and 240 h.Each sample was diluted to make four concentrationsranging from 2.5 to 40 g ml1, and brought to avolume of 100 ml. Groups of 20 s to third instar Ae.aegypti larvaewere added to each sample. Larvaewerefed a powder diet every other day, and mortality wasrecorded at 72 h. A HOBO Pro Series WeatherproofLogger (Onset, Bourne, MA) was used to record theair and water temperaturesof the shade sample. In theshade treatment, air and water temperature ranged

from 23.5 to 40C and 24 to 33.3C, respectively. Fourcontainers with an equivalent amount of DMSO wereset in both shade and laboratory conditions, whichserved as the negative controls. The LC

50, heteroge-

neity factor (Hf), and 95% condence intervals werecalculated using Polo Plus logit probit software(LeOra Software, Petaluma, CA). The proportion oforiginal toxicity was calculated as described in Perezet al. (2007).

Persistence of Biological Activity of-Mangostin inSunlight.Initial experiments found that -mangostinexposed to sunlight for 6 h had reduced larvicidalactivity. Because of -mangostins photosensitivity,the experimental design was modied to capture itsshort half-life. Four concentrations (2.5, 5, 10, and 20g ml1) were prepared in 20-L buckets lined withhigh-density molded polyethyleneinserts(AssociatedBag, Milwaukee, WI) with a total volume of 5 L. Theseexperiments took place on 5 and 6 August at CMAVE.Sky cover was clear on both days (http://www.srh.noaa.gov/jax/f6/KGNV/F6_KGNV_AUG2008). Theambienttemperaturerangedfrom 20.6 to33.9C and 21.7to 34.4C on 5 and 6 August, respectively (http://www.srh.noaa.gov/jax/f6/KGNV/F6_KGNV_AUG2008).Containers were set outside between 9:00 and 10:00

a.m. and were kept under direct sunlight for the re-mainder of the experiment. Samples were taken attime 0 (initial), 45 min, 90 min, 180 min, and 360 min.The initial sample was taken before the containerswere set outside to eliminate any effects of sunlight.Twenty second to third instar Ae. aegypti(Orlando)larvae were added directly to 100-ml samples taken attime 0 (initial), 45 min, 90 min, 180 min, and 360 min.On 5 August, the experiment was set up with tworeplicates, whereas it was set up with three replicateson 6 August. The number ofliving and dead larvae wascounted after 72 h of exposure. Polo Plus probit and

logit analysis software (LeOra Software, Petaluma,CA) was used to calculate the LC50

and Hf. Theproportion of initial toxicity was calculated as de-scribed in Perez et al. (2007).

Assays for Enzymatic Activities in Larvae. Twentylate third instar Ae. aegypti (Rockefeller) were pre-

March 2010 LARSON ET AL.: BIOLOGICALACTIVITY OF-MANGOSTIN AS ALARVICIDE 251


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exposed to 100 ml of water containing 2.5g ml1 of-mangostin for 24 h. This dose was chosen because itwas predetermined as the 72-h LC

50. DMSO equiva-

lents served as the negative control. The larvae werewashed twice in ddH

20 before being used for testing.

After the 24-h induction period, the larvae molted tothe fourth instar, and surviving larvae were used for

biochemical tests. The mortality was 15% after 24-hexposure to 2.5 g ml1 of-mangostin.

Cytochrome P450 Activity. Living larvae exposed to-mangostin or DMSO were cleaned in ddH

20 and

dissected. Larvae were placed in 50 mM sodium phos-phate buffer (pH 7.2) and chilled before dissection.The heads, last abdominal segment, and digestive sys-tem were removed from the larvae. Carcasses werethen used for determining the cytochrome P450 ac-tivity. Cytochrome P450 assays were based on themeasurement of ethoxycoumarin-O-de-ethylase ac-tivity in the body walls. The methods of Boyer et al.

(2005) and De Sousa et al. (1995) were modied todetermine the effect of -mangostin on the cyto-chrome P450 activity in Ae. aegypti larvae. Black,round-bottom 96-well microplates (Catalog 3356,Corning Glass, Corning, NY) were lledwith 100l ofa 0.4 mM 7-ethoxycoumarin solution containing 50mM sodium phosphate buffer (pH 7.2). Individuallarval carcasses were placed in each well and incu-bated for 4 h at 30C. The reaction was stopped withthe addition of 50l of glycine buffer (1 mM, pH 10.4)and 50 l of ethanol. Larval carcasses remained at thebottom of the well and were not removed beforereading. Six wells containing 100 l of phosphatebuffer, 50 l of glycine buffer, and 50 l of ethanolserved as the blank. The uorescence of the reactionmedium was measured from the top of the wells usinga Synergy HT microplate reader (Bio-Tek Instru-ments, Winooski, VT) with 400 nm excitation and 480nm emission lters. The production of 7-hydroxycou-marin (7-OH) was expressed as mol 7-OH/mg lar-vae/min.

Esterase Activity.The methods of Rodriguez et al.(2001) were modied to determine the effect of-mangostin on the esterase activity of fourth instar

Ae. aegypti (Rockefeller) larvae. Individual larvae

were homogenized using a micropestle in a 1.5-mlEppendorf microcentrifuge tube containing 200 l of50 mM sodium phosphate buffer (pH 7.5) before be-ing centrifuged at 13,200 rpm for 15 min. A total of 20l of the supernatant was added to each well of a96-well clear, radius edge, polystyrene microplate(Labsystems, Helsinki, Finland) before 160 l of 0.5mM-naphthyl acetate was added. The reaction wasincubated for 10 min at 30C before it was stoppedwith the addition of 20 l of Fast Blue B salt 0.3%containing 3.5% sodium dodecyl sulfate. A previousstudy using the Rockefeller strain ofAe. aegypti larvae

under similar experimental conditions determined thesaturation of concentration for-naphthyl acetate tobe 70 mM and the optimal reaction time to be 10 min(Rodriguez et al. 2001). The reaction was allowed tocontinue at room temperature for 10 min before theabsorbance was read at 600 nm using a VersaMax

microplate reader (Molecular Devices, Sunnyvale,CA). This assay was performed in replicates of 12 andrepeatedthreetimes (n 36). Protein concentrationswere determinedusing an albumin standard andbicin-choninic acid protein assay kit (Thermo Scientic,Rockford, IL). The esterase activity was expressed asmol/mg protein/min substrate hydrolyzed.

GST Activity.GST activity samples were preparedaccording to the method of Rodriguez et al. (2001).Individual fourth instar larvae were homogenized in200l of 50 mM sodium phosphate buffer (pH 7.2)before being centrifuged at 15,700 gat 4C for 15min. The Sigma-Aldrich(St. Louis, MO) GST assay kitwas used to measure the conjugation ofthe thiol groupof glutathione to the 1-chloro-2, 4-dinotrobenzene(CDNB) substrate. A total of 20l of homogenate wasadded to eachwell before180l of solution containingDulbeccos phosphate buffer (Sigma-Aldrich, St.Louis, MO), glutathione reduced (4 mM), and CDNB

(2 mM). Rodriguez et al. (2001) determined the sat-uration concentration of CDNB to be 50 mM and theoptimum time for reading to be 3 min. The 96-wellat-bottom UV microplate (Corning Glass, Corning,NY) was immediately loaded onto a Synergy HT mi-croplate reader (BioTek, Winooski, VT). After a 1-minlag time, the absorbance was read at 340 nm and thesamples were read every 60 s for 3 min. Protein con-centrations were determined using an albumin stan-dard andbicinchoninic acid protein assay kit (ThermoScientic, Rockford, IL). The activity of GST wasexpressed as mol/mg protein/min substrate conju-

gated.Synergistic Effect of -Mangostin on Temephos

Toxicity Against Mosquito Larvae. Third instar Ae.aegypti(Rockefeller) larvae were used to investigatepotential synergistic effects of-mangostin. Bioassayswere performed in which temephos was serially di-lutedto six concentrations(0.008 0.047)witha 100mltotal volume and 20 larvae were transferred to eachcontainer. A 1 gml1 -mangostin solution wasadded to an identical treatment of temephos. After24 h, the number of living larvae was recorded. Thecontrol consisted of six identical plastic containers

lled with a 1 g ml1

concentration of-mangostin,which resulted in 5% mortality within 24 h.

Statistical Analysis. Probit analysis for all laboratorybioassays was carried out using StatPlus:mac software(AnalystSoft, Vancouver, British Columbia, Canada).The LC

50, LC

5095% condence intervals, and 95%

lethalconcentration (LC95

) were calculatedusing thissoftware. This software package does not calculatecondenceintervals for the LC95, and these values arenot listed as a result. The Hf was calculated by dividingthe2 value by degrees of freedom. StatPlus:mac soft-ware (AnalystSoft, Vancouver, British Columbia, Can-

ada) was also used to perform Student ttests to de-termine the signicance of the enzyme activitiesbetween treatments. Polo Plus logit probit software(LeOraSoftware,Petaluma,CA)wasusedto calculatethe LC

50, condence interval, and Hf in the semield

portion of this study.



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The body weights of the rats were compared for thedifferent treatment groups at 7, 14, 21, and 28 d aftercompound administration began. Each time point wasrst analyzed using Bartletts test to conrm that vari-ance between the groups was uniform. A one-wayanalysis of variance was then conducted for each timepoint. If the analysis of variance showed signicantdifferences between the groups (P 0.05), a Dun-

netts test was performed to determine which groupsdiffered signicantly from one another (P 0.05).

To compare the rats serum levels of ALT betweenthe treatment groups, a repeated measure analysisusing the mixed procedure in SAS (SAS Institute,Cary, NC) was performed. If a signicant difference(P 0.05) between the dosages was found, a pairwisetest (with Bonferroni correction) was performed todetermine which doses were signicantly differentfrom one another (P 0.05).

Results and Discussion

Toxicity of-Mangostin Against Larval Mosquitoes.Third instar larvae of six different mosquito specieswere used to evaluate the toxicity of-mangostin. Theresults for the 72-h toxicity are shown in Table 1. Cx.pipiens pipiensand Cx. pipiens quinquefasciatus werethe most susceptible to-mangostin with an LC

50of

0.84 (0.810.89)g ml1 and 1.6 (1.02.1) g ml1,respectively.An. gambiaewas the least susceptible to-mangostin with a 72-h LC50 of 2.9 (1.85.5)g ml

1

and a LC95

of 12.2 g ml1. SCPI-1, a synthetic SCPI,was evaluated previously against third instar larvae of

An. gambiae andCx. pipiens (Larson et al. 2008). In the

previous study, the 72-h LC50of SCPI-1 was 6.5 3.0g ml1 and 2.4 0.8g ml1 inAn. gambiaeandCx.pipiens,respectively (converted fromM, molecularweight of SCPI-1 459.19 d). Compared with SCPI-1,-mangostin is more effective againstAn. gambiae andCx. pipiens by a factor of 2.2 and 2.9, respectively(SCP11 LC

50/-mangostin LC50). Interestingly,An.

gambiae was the least susceptible to both SCPIs,whereasCx. pipienswas the most susceptible. Thereare two possible explanations for the relative species-specic susceptibility to SCPIs. First, it is possible thatthere may be slight structural differences between the

SCP-2 of Cx. pipiens andAn. gambiae, which couldresult in differences in afnity for cholesterol andSCPIs (Larson et al. 2008). Second, exposure to SCPIsmay induce higher levels of detoxication enzymes in

An. gambiaecompared withCx. pipiens.Further stud-ies are needed to test these possible mechanisms.

The 72-h LC50

inAe. aegyptiranged from 2.2 to 2.5g ml1 depending on the strain tested. The Rock-efeller and Orlando strain ofAe. aegypti had similarsusceptibilitywith LC50valuesof 2.2(1.922.6) (Table1) and 2.5 (2.32.7) gml1 (Table 2), respectively.OurLC

50values weremuch lower thanthose found by

Ee et al. (2006). They tested-mangostin against thirdinstar Ae. aegypti larvae and reported a 24-h LC

50of

19.1 gml1 (Ee et al. 2006). However, the 7.6-folddifference in LC

50value ndings can be attributed to

the differences in duration between the studies. The24-h bioassays performed by Ee et al. (2006) werebiased toward faster acting chemicals. Our previousstudy suggests that SCPIs are slow acting; therefore,evaluation of SCP-2 inhibitors should be performedover 72 h (Larson et al. 2008).

Continuous Versus 24-h Exposure to -Mangostin.Although previous studies suggest that SCPIs are slowactingand shouldbe evaluated for 72 h, it is not knownwhether continuous 72-h exposure to SCPIs is neededfor effective control of mosquito larvae (Larson et al.2008). In the current study, we exposed Ae. aegypti(Orlando) larvae to -mangostin for 24 h and com-pared thetoxicity to larvaethat underwentcontinuousexposure to-mangostin. It was found thatAe. aegyptilarvae exposed to-mangostinfor 24 h had a 72-h LC

50

of 2.7 (2.33.3)gml1, whereas larvae exposed con-tinuously had a 72-h LC

50of 2.5 (2.32.7) g ml1

(Table 2). The 95% condence intervals of the twotreatments overlap, which suggests that the differencebetween treatments is not signicant at a 5% level.

Thus, 24-h exposure to -mangostin had detrimentaleffects onAe. aegyptilarvae that did not immediatelyresult in mortality.

After 24-h exposure to -mangostin (2.5gml1),large differences were also observed in the total sol-uble protein and mass of the body wall. The concen-tration of soluble protein from control larvae was 0.41(0.360.45) mg ml1 per larva, whereas the total sol-ubleprotein from larvaetreated with-mangostin was0.17 (0.150.2) mg ml1 per larva (Table 3). This isconsistent with the average mass of larval body walltaken from 50 control larvae and 50 larvae exposed to

2.5 gml1

of -mangostin. Larval body walls dis-sected from larvae that were exposed to DMSO and-mangostin weighed 0.92 and 0.56 mg, respectively(Table 3). There was a greater relative differencebetween control and treated in amount of solubleprotein (2.4-fold) compared with the difference of

Table 1. Seventy-two-hour toxicity assay of -mangostinagainst third instar larvae of six mosquito species

Species n LC50gml

1

(95% CL)LC95

gml1Slope(SE)

Hf

Ae. aegypti(Rockefeller) 5 2.2(1.92.6) 5.7 3.9 (0.4) 2.50An. gambiae 4 2.9(1.85.5) 12.2 2.7 (0.6) 5.05An. quadrimaculatus 11 1.7 (1.51.9) 2.7 8.1 (1.4) 0.42

An. stephensi 6 1.9 (1.62.4) 8.1 2.6 (0.3) 1.46Cx. pipiens pipiens 6 0.84(0.80.9) 2.0 4.3 (0.2) 1.71Cx. p. quinquefasciatus 8 1.6 (1.02.1) 4.2 3.8 (1.1) 3.93

Table 2. Continuous versus 24-h exposure on the72-h toxicityin third instar Ae. aegyptilarvae

Exposuretime

n LC50gml

1

(95% CL)LC95

gml1Slope(SE)

Hf

Continuous 6 2.5 (2.32.7) 4.3 7.2 (0.8) 0.0324 h 6 2.7 (2.33.3) 5.2 5.9 (0.9) 2.75

Third instarAe. aegypti(Orlando) larvae were exposed to -man-gostin for 24 h and continuously. In the 24-h treatment, larvae wereremoved from -mangostin solutions and washed twice before beingtransferred to ddH

20 for the remainder of the experiment (see Ma-

terials and Methods).



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larval body wall masses between the control and

treated (1.6-fold). Work performed by Kim et al. (per-sonal communications) suggests that -mangostin is afeeding deterrent on the Colorado potato beetle. It ispossible that -mangostin treatment led to decreasedfeeding and lipid metabolism in the larvae, whichresulted in lowered body mass compared with thecontrol. The Bradford method (Bradford 1976) of de-termining protein concentration cannot discriminatebetween protein from dietary sources in the midgutand protein from the insect body.-Mangostin may bea feeding deterrent for mosquito larvae and couldaccount for the relatively large differences in solubleprotein. Nonetheless, the presence of -mangostindrastically reduced the total soluble protein and av-erage mass of larval bodies after only 24 h of exposure,whereas the 24-h mortality was 15%.

Toxicity of-Mangostin Against Adult Mosquitoes.-Mangostin was applied topically to 5- to 7-d-oldadultAe. aegypti(Orlando) to test its potential use asan adulticide. The results of this biological assay aresummarized in Table 4. The 48-h 50% lethal dose(LD

50) was 2.2 (2.02.4) %wt:vol (mg compound/100

l solvent), which is within the same order of mag-nitude as the LC50values found from bioassays per-formed against the rice weevil (S. oryzaeL.) and the

brown plant hopper (N. lugensStal.) (Bullangpoti etal. 2004, 2006). Compared with the LD

50valuesagainst

mosquito larvae and commercially available insecti-cides, the LD

50values for topically applied-mangos-

tin are several orders of magnitude higher. For in-stance,Pridgeon et al.(2008) evaluated19 insecticidesagainst adultAe. aegyptiusing similar protocol to theone used in this study. It was found that Ae. aegyptiwere most susceptible to Fipronil with a LD50 of 4.6107g insecticide/mgmosquito andleast susceptibleto Bifenazate with a LD

50 of 1.5 g insecticide/mg

mosquito (Pridgeon et al. 2008). Fourteen of the 19compounds tested had LD50values 10

2 g insec-ticide/mg mosquito. When the LD

50of-mangostin is

converted to the same units described in Pridgeon etal. (2008) (using the average weight of 7-d-old female

Ae. aegypti 2.85 mg and 0.5 l of compound perfemale), the LD

50 of-mangostin would be 4 g

insecticide/mg mosquito. Therefore, it can be con-cluded that -mangostin has limited adulticidal prop-erties compared with many commercial products.

There are several possible explanations for the lim-ited activity of-mangostin in adultAe. aegypti.First,the extreme hydrophobicity of -mangostin mayhinder its ability to penetrate the cuticle. Second,-mangostin may need to be ingested to be effectiveagainst insects because Aedes sterol carrier protein-2(AeSCP-2) is highly expressed in the midgut (Krebsand Lan 2003). Third, SCPIs may not be effectiveagainst adult mosquitoes because AeSCP-2 is nothighly expressed in the pupal and adult stages (Krebsand Lan 2003).

Persistence of Biological Activity of-Mangostin inSemifield Conditions. When -mangostin was ex-posed to direct sunlight, the initial (time 0), 45-min,and 90-min samples tested against third instar Ae.aegypti larvae (Orlando) produced LC

50 values of

4.9 (4.25.7), 10.3 (9.511.2), and 21.8 (18.228.6)gml1, respectively (Table 5). LC50values from the

initial time point (0 h) were approximately 2-foldhigher than the 72-h LC

50determined in laboratory

assays (Table 2). These differences may be caused bythe different containers usedbetween studies.-Man-gostin is extremely hydrophobic, which might cause itto be more attracted to the liners used in the semieldassays than the plastic containers used in the labora-tory assays. The LC

50values for the 90-min sample

were interpolated because two of the ve samples didnot produce at least 50% mortality. There was nomortality detected beyond 90-min time points, andLD

50 values could not be obtained after this time

point. To determine the proportion of initial toxicityremaining, the LC50

values for each time point weredivided by the LC

50value for the initial time point (0).

These values were plotted over time. As predicted, thepersistence had a negative linear response to sunlight.It was determined that-mangostin has a half-life of

Table 3. Soluble protein concentration and body-wall mass

Mean (95% CL) Pvalue

Total soluble protein(mg/ml) (n 56)

Control 0.41 (0.360.45) 0.001-Mangostin treated 0.17 (0.150.2)

Average mass of body wall

(mg) (average of 50)Control 0.92 NA-Mangostin treated 0.56

Late third instar larvae of Ae. aegypti (Rockefeller strain) wereexposed to 2.5 gml1 of-mangostin for 24 h. Early fourth instarlarvae were collected to determinesoluble protein concentration andmass of fat bodies. Studentt tests were used to determinePvalues.Average mass of body walls was calculated by weighing 50 carcassesand dividing their total weight by 50. Therefore, Pvalues are notapplicable to the average mass of body walls.

Table 4. Forty-eight-hour toxicity assay of -mangostinagainst 5- to 7-d-old adult Ae. aegypti (Orlando strain)

LD50

(%wt:vol) LD90

(%wt:vol) Slope (SE) Hf

2.2 (2.02.4) 10.1 2.5 (0.1) 1.45

Table 5. Persistence of-mangostin exposed to direct sunlight

Exposureto sunlight

LC50gml1

(95% CL)Slope(SE)

HfProportion of

original toxicityremaining

0 4.9 (4.25.7) 3.2 (0.3) 1.73 1145 min 10.3 (9.511.2) 4.6 (0.4) 1.05 0.4890 min 21.8 (18.228.6) 2.7 (0.4) 1.40 0.22

Half-life52.8 min

Equation:y 0.0086x 0.9544 R2 0.96

-Mangostin was exposed to direct sunlight, and samples fromvarious time points were tested againstAe. aegypti(Orlando) larvae.Proportion of original toxicity was calculated by dividing the 72-hLC50 of each time point by the LC50 corresponding to the initialsample (time 0).There wasno mortalitydetected beyond90-min timepoints, and LD50values could not be obtained after this time point.



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52.8 min with full sun exposure (Table 5). Relative tomost commercially available pesticides, -mangostinis very photosensitive. In the post Silent Springeraof pest management, environmental degradation ofinsecticides is a positive attribute (Carson 1962).However, insecticides must persist in theenvironmentlong enough for effective control. Formulation tech-

nology may also help photosensitive compounds over-come environmental degradation. For example,methoprene has a half-life of 3040 h depending onthe initial concentration (Schooley et al. 1975). Slowrelease formulations combined with UV-blocking ad-ditives allow some formulations of methoprene to re-main biologically active in the environment formonths. Thus, the photosensitivity of -mangostincould potentially be corrected with existing formula-tion technology.

The persistence of-mangostin was also evaluatedin the laboratory and shade. Data were not shown for

the shade and laboratory treatments, because -man-gostin precipitated out of solution in these treatments.-Mangostin is practically insoluble in water (MerckIndex 1989), which suggests that it dissociated withthe carrier, DMSO. However, further studies areneeded to determine the mechanism of the precipi-tation of-mangostin stored in the shade and labora-tory.

Biological Effects of-Mangostin on Rats. Mamma-lian SCP-2s have low amino acid sequence identity tothe mosquito SCP-2 (Krebs and Lan 2003); however,the three-dimensional structures show very high ho-mology, with root mean square (rms) difference be-tween 97 structurally equivalent amino acids only 1.15 (Dyer et al. 2003). Whether SCPIs such as -man-gostin would affect the function of vertebrate SCP-2is unknown, although vertebrate SCP-2 seemed to benonessential for mammalian survival (Kannenberg etal. 1999, Fuchs et al. 2001). To test the toxicity of-mangostin in mammals, young male rats were fedvaried dosages of -mangostin for 4 wk, duringwhich body weight and a biomarker for hepatocytedamage were measured. Survival rates for treated andcontrol animals were 100%. During daily monitoring,no rats were found to be dehydrated, lethargic, un-

responsive, or excessively stressed. The Bartlett testdemonstrated that variance of body weight betweenthe groups was uniform for each time point(P0.47).There was no signicant difference in body weightbetween the groups at any of the given time points(day 7,P 0.709; day 14,P 0.797; day 21,P 0.808;day 28,P 0.672). The result of our toxicity assays inthe young rats is consistent with the report on adultrats (Sornprasit et al. 1987).

Serum ALT levels differed signicantly betweentime points (P 0.0002), with levels of the enzymedecreasing in the bloodstream as the animals aged.

However, there was no signicant difference in serumALT levels between groups (P 0.5007) or when timepoint and dose were analyzed simultaneously (P0.9463), suggesting that-mangostin did not result inhepatocyte damage that facilitated the release of ALTinto the bloodstream. The results of this preliminary

toxicology study of-mangostin in young rats suggestthat-mangostin exhibited little acute adverse effectin mammals, which is consistent with the observationsin SCP-2 knockout studies that show that the verte-brate SCP-2 is not essential for survival (Kannenberget al. 1999, Fuchs et al. 2001).

Enzymatic Responses of Fourth Instar Mosquito

Larvae to -Mangostin. In response to xenobiotics,insects must break down the chemical into solubleform before they can excrete it. To perform thesetasks, insects often rely on three superfamily of en-zymes: cytochrome P450 monooxygenases, esterases,and GST (Brogdon and McAllister 1998). Therefore,when exposed to a xenobiotic, enzyme activities areoften induced to higher levels (Brattsten et al. 1986).The reasons for measuring cytochrome P450, esterase,and GST activities in the current study are 2-fold. First,these enzymatic activities were measured to investi-gate which enzymes are responsible for detoxifying

-mangostin. Second, enzyme activities were alsomeasured to assess reports that-mangostin inhibiteddetoxication of enzymatic activities. Studies in Thai-land with the rice weevil (S. oryzae L.) and the brownplant hopper (N. lugensStal.) suggest that-mangos-tin inhibits esterase, acetylcholinesterase, carboxyles-terase, and GST activities (Bullangpoti et al. 2004,2006). On one hand, these ndings are unexpectedbecause -mangostin is extremely hydrophobic anddetoxication of enzyme activities is usually tempo-rarily induced to higher activities by the introductionof foreign lipophilic compounds (Brattsten et al.1986). However, it has been demonstrated that someplants have evolvedto producesecondary metaboliteswith synergistic effects to enhance their chemical de-fense (Berenbaum andNeal 1985). Nonetheless, theseclaims need to be re-evaluated to determine whether-mangostin has synergistic properties.

As expected, cytochrome P450 and GST activitiesincreased signicantly whenAe. aegypti (Rockefeller)larvae were exposed to 2.5 g ml1 of-mangostin for24 h, which suggests that these enzymes play a majorrole in its detoxication. The mean cytochrome P450activity of the control and treatment were 6.4 0.36and 8.4 0.6mol 7-OH/mg larvae/min, respectively

(Table 6). This is the rst report on cytochrome P450activities in response to -mangostin exposure in in-sects.

In this study, the activity of GST increased by afactor of 1.25 in treated larvae (Table 6). Our ndingscontradict the previous claims that -mangostin in-hibits GST in insects. In studies usingN. lugens,GSTactivity decreased up to 3-fold after 24-h exposure to5.44% (wt:vol) (24-h LD

50)-mangostin (Bullangpotiet al. 2006). The differences between studies may bebecause of species-specic response to -mangostin.

Esterase activities decreased in response to -man-

gostin,although it hadonly moderate reduction of 12%(P 0.05). The respective esterase activity of thecontrol and treatment was 0.26 0.01 and 0.23 0.01mol -napthol/mg protein/min (Table 6). Thesendings are consistent with the claims that-mangos-tin inhibits esterase activity by 1.2-fold in S. oryzae



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(Bullangpoti et al. 2004). In an attempt to determine

whether -mangostin has synergistic effects, 1 gml1 of-mangostin was added to six concentrationsof temephos and compared with the toxicity of anequal treatment of temephos without -mangostin.Temephos was chosen as an insecticide because it isknown that esterase aids in the detoxication of or-ganophosphates (Hemingway and Ransom 2000). In-terestingly, we found that -mangostin decreased theLC

50of temephos by a factor of 1.6. The LC

50of larvae

exposed to -mangostin and temephos was 0.012(0.0110.013) g ml1, whereas the LC50 of temephosalone was 0.019 (0.0160.023) g ml1 (Table 7).Exposure of-mangostin for 24 h at this concentration

wasnonlethalto third instar larvaeas themortalitywas5%. Although the addition of 1 gml1 of-man-gostindecreased theLC

50of temephos, it is not known

whether these effects are synergistic or additive. Itwould be interesting to repeat this experiment usingtemephos-resistant mosquitoes, as synergistic effectsmay be more apparent in a resistant strain.

In summary,-mangostin is a novel insecticide thatis toxic to larvae of several mosquito species. Its novelmode of action would be a welcome addition to thelimited vector control tool box.Considering its lowmammalian toxicity, it may ll a nicheas anAe. aegypti

larvicide in storage containers forpotable water.Com-pared with commercially available larvicides, such astemephos, -mangostin is2 orders of magnitude lesstoxic. However, formulations that can deliver -man-gostin to the midgut where AeSCP-2 is highly ex-pressed may improve its efcacy. Thus, future studiesshould aim at delivering -mangostin to the midgut as

well as increasing environmental stability through im-provements in formulation technology.

Acknowledgments

We thank Drs. Bruce Christensen and Susan Paskewitz forproviding the mosquito eggs, and David H. Dyer for histechnical support. This work was supported by the U.S. NavyEntomologyHealth ServicesCollegiate Program (HSCP)(toR.T.L.); the University of Wisconsin College of Agriculture;Grant W9113M-051-0006 from the Deployed War FighterProtection Research Program, which was administered bythe U.S. Armed Forces Pest Management Board of the U.S.Department of Defense; and National Institutes of HealthResearch Grant 5R01AI067422 (to Q.L.).

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Treatment LC50gml

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gml1Slope(SE)

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Temephos 0.019 (0.0160.023) 0.041 4.8 (0.8) 2.07Temephos -mangostin

0.012(0.0110.013) 0.023 6.2(0.6) 0.79

Third instarAe. aegypti(Rockefeller) were used. The temephos -mangostin treatment contained 1 gml1 of-mangostin for 24 h(seeMaterials and Methods).



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Received 22 June 2009; accepted 21 December 2009.


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