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Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf
This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.
Biocontrol Science and Technology (2002) 12, 7- 17
Mortality in Three African Tephritid Fruit Fly Puparia andAdults Caused by the Entomopathogenic Fungi, Metarhizium
anisopliae and Beauveria bassiana
S. EKESI, N. K. MANIANIA and S. A. LUX
International Centre of Insect Physiology and Ecology (ICIPE), PO Box 30772,Nairobi, Kenya
(Received for publication 6 February 2001; revised manuscript accepted 16 May 2001)
The pathogenicity of 13 isolates of Metarhizium anisopliae and two isolates of Beauveriabassiana to Ceratitis capitata and Ceratitis var. rosa fasciventris exposed as late third instarlarvae in sand was evaluated in the laboratory. All isolates caused a signiWcant reduction inadult emergence and a corresponding large mortality on puparia of both species. All isolatesalso induced large deferred mortality in emerging adults following treatment as late third instarlarvae. On C. capitata , seven isolates (M. anisopliae ICIPE 18, 20, 32, 60 and 69 andB. bassiana ICIPE 44 and 82) caused signiWcantly higher mortality on puparia than otherisolates. With the exception of ICIPE 32, the other four isolates of M. anisopliae above werethe most pathogenic against C. r. fasciventris. Dose-response study carried out with theseisolates of M. anisopliae on the two species of Xies above plus another species, Ceratitis cosyrashowed that the dose-mortality regression lines of ICIPE 18 and 20 were steeper with lowerLC50 values when compared with ICIPE 60 and 69 on the three species. When these twoisolates were evaluated with regard to their pathogenicit y to diVerent pupal age, adult emergencewas found to increase with increasing pupal age with a corresponding decrease in mortality inpuparia and emerging adults in the three species of fruit Xies. M. anisopliae ICIPE 18 and 20were equally pathogenic to all pupal ages tested in C. capitata and C. cosyra but ICIPE 18was more pathogenic to older puparia of C. r. fasciventris than ICIPE 20. Our results suggestthat soil inoculation with M. anisopliae under mango trees might form an important componentof integrated pest management strategies in areas where these three species of fruit Xy coexist.
Fruits constitute a major agricultural commodity in Africa with export targeted towardslarge markets in Europe and the Middle East. Mangoes, guava, coVee, papaya, citrus, appleand avocado are among the commonest fruits grown for export. Three tephritid fruit ¯ies,
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8 S. EKESI ET AL.
Ceratitis capitata (Weidemann), Ceratitis var. rosa fasciventris (Bezzi) and Ceratitis cosyra(Walker) (Diptera: Tephritidae), however, constitute a serious threat to fruit production inAfrica. In Kenya alone, about 90 000 tons of mangoes are produced annually but anestimated 20- 40% of the fruit is lost to fruit ¯y infestation at the time of ripening. Lossesat the orchards of professional producers ranged between 10- 25% while small holders losebetween 30- 80% (Lux et al., 1998). In addition to these direct losses, producer countriesmay also lose potential markets due to stringent quarantine regulations imposed byimporting countries to avoid entry and establishment of unwanted fruit ¯ies. In most partsof Africa, the small holder farmers, which are responsible for producing the bulk of thefruits, rarely apply any control measures against fruit ¯ies resulting in large economic losses.It is, however, most likely that application of an appropriate control method could enhanceproduction of high quality fruits for domestic urban markets in addition to ensuring thatstringent quarantine and quality regulations for export markets are met.
During development, third instar larvae of most fruit ¯y species drop from fruits to theground, burrow into the soil and form a puparium (White & Elson-Harris, 1992). Animportant part of a fruit ¯y suppression and eradication programme therefore includes soiltreatment with insecticides beneath host trees to kill fruit ¯y larvae and puparia (Mohamadet al., 1979; Saul et al., 1983; Roessler, 1989; CDFA, 1993). Currently, the organophosphat einsecticide diazinon is the most widely used soil insecticide against fruit ¯y larvae/puparia.The use of synthetic insecticides, including diazinon for pest control is, however, associatedwith various ecological problems such as environmental contamination, adverse eVects onnon-target organisms and the development of resistance (Croft, 1990). Additionally, thepersistence of diazinon in the soil is known to decrease within 2 weeks, therefore, requiringrepeated application and its future use in fruit ¯y eradication and suppression programmeshas been strongly questioned (Roessler, 1989).
Although entomopathogeni c fungi are known to attack fruit ¯y species (White & Elson-Harris, 1992), very little has been done to exploit these pathogens for fruit ¯y management.Puparia of rose fruit ¯y Rhagoletis alternata Meigen are attacked by the fungus Scopulariopsisbrevicaulis (Sacc.) Bainier (Lipa et al., 1976). Various isolates of Metarhizium anisopliae(Met.) Sorok. and Paecilomyces fumosoroseus (Wize) Brown and Smith were found to bepathogenic to adult C. capitata and infection was reported to reduce fecundity and fertility(Castillo et al., 1999). Since most entomopathogenic fungi are soil-borne microorganisms,their incorporation into the soil targeted at pupariating larvae and puparia can form animportant component of an integrated pest management strategy for fruit ¯ies. Numerousstudies have demonstrated the success of soil treatment with fungal pathogens for the controlof diVerent agricultural pests (Watt & Le Brun, 1984; Gaugler et al., 1989; Krueger et al.,1991; Zimmermann, 1994; Rath et al., 1995; Booth and Shanks, 1998). Here we report theresult of studies that evaluated the eVect of sand treatment with 13 isolates of M. anisopliaeand 2 isolates of Beauveria bassiana (Bals.) Vuill. on late third instar larvae, puparia andemerging adults of C. capitata, C. r. fasciventris and C. cosyra. Due to lack of insects,C. cosyra was not included in the initial experiments but was added in the dose responseand pupal age experiments. The intent of the study was to select an isolate that is eVectiveagainst the three species of ¯ies since this will be more attractive from a commercialstandpoint than strict speci®city to one species of fruit ¯y.
MATERIALS AND METHODS
InsectsLarvae of the three fruit ¯y species (C. capitata, C. r. fasciventris and C. cosyra) were frommass rearing stock maintained at ICIPE. The larvae were reared on a carrot and sugarbased arti®cial diet (Hooper, 1987) and adult ¯ies were maintained on a mixture of sugar(3 parts) and commercial enzymatic yeast hydrolysate (1 part) based arti®cial diet.
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PATHOGENICITY OF ENTOMOPATHOGENIC FUNGI TO FRUIT FLIES 9
TABLE 1. Origin of fungal isolates tested against the three species of fruit ¯ies
Date ofFungal species Isolate Host species (family) Substrate Locality (country) isolation
FungiThe 15 fungal isolates used in our experiments (Table 1) were obtained from the ICIPEmicrobial bank except M. anisopliae var. acridum (IMI 330 189) registered as Green Musclewhich was donated by LUBILOSA (French acronym for Lutte Biologique contre les Locusteset les Sauteriaux). The fungi were grown on Sabouraud dextrose agar (SDA) in Petri dishesand maintained at ambient temperature (22- 28ëC) in complete darkness.
Preparation of Conidial SuspensionConidia were harvested from 2- 3 week old surface cultures by scraping. Spores weresuspended in 20 ml sterile distilled water containing 0.05% Triton X-100 in glass bottlescontaining 3 mm glass beads. Bottles were stoppered and vortexed for 5 min to produce ahomogeneous conidial suspension. Conidia were then quanti®ed with a haemocytometerfollowing serial dilution in sterile distilled water. Viability of conidia were determined byspread-plating 0.1 ml of conidial suspension (titrated to 3 3 106 conidia ml- 1 ) on four SDAplates. A sterile cover slip was placed on each plate and incubated in complete darkness atambient temperature. Percentage germination was examined after 24 h from 100-sporecounts on each plate.
Inoculation of InsectsDried beach sand (50 g; 99% sand, 0.5% silt, 0.5% clay, pH 5.2, obtained from the shoresof Lake Victoria, western Kenya) were sifted through a 16-mesh screen and transferred into90-mm diameter Petri dishes. A standard concentration of 1 3 108 conidia ml - 1 (5 ml) wasused to inoculate the 50 g lots of sand and which were then vigorously mixed with a spatula.Control lots were treated with sterile distilled water containing 0.05% Triton X-100. Twentymature third instar larvae of C. capitata and C. r. fasciventris that were ready to pupatewithin the next 24 h were then introduced into each Petri dish for pupation and the disheswere placed inside a humid transparent plastic container and maintained at ambienttemperature (25- 28ëC) under a photoperiod of L 12: D 12. Seven days after the introductionof the larvae, puparia were removed from the treated sand and transferred into anotherPetri dish containing untreated sand and placed in Plexiglass cages (15 3 15 3 20 cm). Acotton bud soaked with water and a small lid (1 3 2 cm) containing a 4:1 mixture of sugarand yeast hydrolysate was introduced in the cage as food source. Test insects were maintainedat the same condition as above. The number of adult ¯ies that emerged from treated andcontrol sand was recorded daily until 14 days after the ®rst emergence. Records were also
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10 S. EKESI ET AL.
kept for the number of puparia that failed to emerge. Adult ¯ies that died during this periodand puparia that failed to emerge were surfaced sterilized in 2% sodium hypochlorid efollowed by two rinses with sterile distilled water and transferred to Petri dishes lined withmoistened ®lter paper. The criteria for scoring mycoses were (1) failure of puparia todevelop accompanied with fungal sporulation on colonized puparia and (2) death of adultsaccompanied with fungal sporulation on the cadavers. Each treatment was replicated fourtimes with 20 insects per replicate.
In a similar set up as above, dose mortality relationship of 4 of the isolates that werehighly virulent to both species were worked out using four doses of inoculum: 1 3 105,1 3 106, 1 3 107 and 1 3 108 conidial ml - 1. In this experiment, C. cosyra was included. Theisolates with the lowest LC5 0 values were then tested against 12, 48 and 96 h old puparia toevaluate the eVect of age on the susceptibility to the selected isolate using a concentrationof 1 3 108 conidia ml- 1. In this experiment, Petri dishes were initially ®lled with treatedsand to the depth of 0.4 cm. Puparia were then placed on the sand and covered with another0.4 cm of treated sand. Treatment evaluation was similar to the one described above. Inboth the dose response and pupal age experiments, four replicates of 20 insects weremaintained for C. capitata and C. r. fasciventris and ®ve replicates of 10 insects per replicatefor C. cosyra for each concentration and pupal age.
Statistical AnalysisPercentage mortality data were corrected for control mortality (Abbott, 1925) and the datawere normalized through angular transformation and then subjected to analysis of variance(ANOVA) followed by mean separation by the Student-Newman-Keuls’ test (P 50.05) usingthe ANOVA procedure. The data on mycosed adults were weighted to compensate fordiVerences in the number of insects per treatment (Sokal & Rohlf, 1981) before subjectingto ANOVA. Regression analysis was used to determine the functional relationship betweenlog concentration of inoculum and probit of mortality (puparia and adult mycosis combined)using the Probit procedure. All analyses were performed using the SAS (1996) package.
RESULTS
In viability tests, germination for all the isolates ranged from 86 to 94%. All pupariatinglarvae treated with fungi pupated normally but infection established in puparia and emergingadults in all the species of fruit ¯ies. In C. capitata, percentage adult emergence was 84% inthe control treatment and varied from 8 to 40% in fungal treated sand (F 528.5; df 515,48;P 50.0001) (Figure 1). Percentage of puparia with visible signs of mycosis ranged from 25to 94% (F 5144.04; df 515,48; P 50.0001) (Figure 1). Isolates ICIPE 32 and 69 were themost pathogenic to C. capitata puparia followed by ICIPE 18, 20, 44, 60 and 62 (Figure 1).Deferred mortality in C. capitata adult ¯ies ranged from 36% to 100% (F 513.36; df 515,48;P 50.0001) (Figure 1) and natural mortality in the control did not exceed 3%.
In C. r. fasciventris, adult emergence was 86% in the control and ranged from 6 to 68%in fungal treated sand (F 517.15; df 515,48; P 50.0001) (Figure 2). The percentage ofmycosed puparia was highest in ICIPE 69 (90%) followed by ICIPE 18, 20 and 60 withmortality of between 83 to 88% (F 554.19; df 515,48; P 50.0001) (Figure 2). Deferredmortality in adults due to mycosis ranged from 13% to 100% in all the isolates (F 528.76;df 515,48; P 50.0001) (Figure 2) and natural mortality in the control was 5%.
Figure 3 shows the plots of the probit of regression estimates for the four isolates for eachof the three species. The results revealed that on the three species, the slope of regression linesfor isolates ICIPE 18 and 20 were steeper than those of ICIPE 60 and 69, indicating that thetarget insects will be more vulnerable over a given time to increasing doses of conidia of theformer isolates than the later ones. The response of ICIPE 69 in C. r. fasciventris and C. cosyrawas highly variable with low slopes (Figure 3) indicating a weak concentration response.Probit lines were not parallel in C. capitata and C. cosyra but a low level of signi®cance
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PATHOGENICITY OF ENTOMOPATHOGENIC FUNGI TO FRUIT FLIES 11
FIGURE 1. Pathogenicity of diVerent isolates of Metarhizium anisopliae and Beauveria bassiana to Ceratitiscapitata: Mean (% 6 SE) adult mycosed (a), puparia mycosed (b), and adult emergence (c)after treatment with a concentration of 1 3 108 conidia ml-1. Bars with the same letter do notdiVer signi®cantly by SNK (P 50.05).
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12 S. EKESI ET AL.
FIGURE 2. Pathogenicity of diVerent isolates of Metarhizium anisopliae and Beauveria bassiana to Ceratitisvar. rosa fasciventris: Mean (% 6 SE) adult mycosed (a), puparia mycosed (b), and adultemergence (c) after treatment with a concentration of 1 3 108 conidia ml-1. Bars with thesame letter do not diVer signi®cantly by SNK (P 5 0.05).
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PATHOGENICITY OF ENTOMOPATHOGENIC FUNGI TO FRUIT FLIES 13
FIGURE 3. Log-probit regressions of mortality caused by four isolates of Metarhizium anisopliae onCeratitis capitata, Ceratitis var. rosa fasciventris and Ceratitis cosyra.
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14 S. EKESI ET AL.
TABLE 2. Susceptibility of pupal stages of Ceratitis capitata, Ceratitis var. rosa fasciventris and Ceratitiscosyra to Metarhizium anisopliae treated at 1 3 108 conidia ml-1
C. capitata C. fasciventris C. cosyraParameter/age (%) ICIPE 18 ICIPE 20 ICIPE 18 ICIPE 20 ICIPE 18 ICIPE 20
Adult emergence, puparia mycosed and natural mortality in adult in the control were 82, 0 and 3%,respectively, for C. capitata; 76, 0 and 0%, respectively for C. r. fasciventris and 78, 0 and 4%, respectively forC. cosyra. Means (6 SE) for each parameter within a column followed by the same letter do not diVersigni®cantly by SNK (P 50.05)
(X2 510.8, df 53, P 50.0419) was obtained with C. r. fasciventris in parallelism chi-square.The LC50 values were: ICIPE 18 (C. capitata: 1.7 3 105, C. r. fasciventris: 2.3 3 105, C. cosyra:3.4 3 106 conidia ml- 1; ICIPE 20 (C. capitata: 5.5 3 104, C. r. fasciventris: 4.6 3 105, C.cosyra: 8.5 3 105 conidia ml - 1 ). The estimated LC5 0 for the other isolates were: ICIPE 60:(C. capitata: 7.4 3 105, C. r. fasciventris: 1.5 3 106, C. cosyra: 4.6 3 106 conidia ml- 1 ); ICIPE69: (C. capitata: 1.6 3 106, C. r. fasciventris: 3.9 3 106, C. cosyra: 7.7 3 106 conidia ml- 1 ).
In the pupal age experiment, adult emergence increased with increasing pupal age with acorresponding decrease in mortality in puparia and emerging adults in all the three speciesof fruit ¯ies (Table 2). ICIPE 18 and 20 were equally pathogenic to the various pupal agesof C. capitata and C. cosyra (Table 2). ICIPE 18, however, appears to be more pathogenicto older puparia (96 h) of C. r. fasciventris than ICIPE 20 (Table 2).
DISCUSSION
The entomopathogenic fungi we used can induce high mortality in puparia of C. capitata,C. r. fasciventris and C. cosyra if pupariating third instar larvae are exposed to sand treatedwith conidial suspension. The overall eVect of this treatment led to a signi®cant reductionin adult emergence in all the three species of fruit ¯ies tested. Out of the 15 isolates evaluatedagainst C. capitata and C. r. fasciventris, seven isolates (M. anisopliae ICIPE 18, 20, 32, 60and 69 and B. bassiana ICIPE 44 and 82) caused signi®cantly higher pupal mortality onC. capitata while four isolates (M. anisopliae ICIPE 18, 20, 60 and 69) were found to behighly pathogenic to C. r. fasciventris. Out of these seven highly pathogenic isolates, fourisolates (M. anisopliae ICIPE 18, 20, 60 and 69) were selected for dose response study basedon the criterion of high pathogenicity to both C. capitata and C. r. fasciventris. This criterionwas found valid since both species occupy the same ecological niche and an isolate with abroad range of activity would be more desirable than strict speci®city to one species. Thedose response study revealed that these four isolates were also highly pathogenic to C. cosyra.
Generally, all species of fruit ¯ies pupated normally but 2 days after pupation, spots ofmelanization were observed on the integument of the puparia (Ferron, 1985). At ®ve daysafter pupation, mycelial growth was observed on colonized puparia followed by profusesporulation on the surface of the puparia (data not shown). A high level of sporulation is
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PATHOGENICITY OF ENTOMOPATHOGENIC FUNGI TO FRUIT FLIES 15
an important function for selected isolates because each puparium dying in the soilconstitutes an infection focus, which will serve as a source of inoculum for infecting otherhealthy pupariating larvae. Additionally, a high level of sporulating puparia occurring inthe soil will ensure that inoculum threshold and contact chances between conidia andhealthy pupariating larvae and puparia is increased thus ensuring that disease is transmittedthrough generations of the pests. It has been reported that B. bassiana hyphae can spreadrapidly from infected weevil cadavers through the soil (Gottwald & Tedders, 1984).
Entomopathogeni c fungi are being developed in various countries for the control ofdiVerent agricultural pests and some products are now available commercially (Lacey &Goettel, 1995; Jackson, 1999). Their safety and selectivity to non-target bene®cial organismsmakes them ideal candidates for integration into various pest management programmes(Goettel & Johnson, 1992; Ekesi et al., 1999). Additionally, their production is easy andcheap and do not require high input technology (Prior, 1988). Our study suggests that soildrenches with entomopathogeni c fungi may be an eVective integrated pest managementcomponent for the control of C. capitata, C. r. fasciventris and C. cosyra in mango orchards.Soil application of entomopathogeni c fungi has been undertaken in various parts ofthe world as a cost-eVective management technique for many insect pests. In Poland,Wojciechowska et al. (1977) showed increased mortality of pupating Colorado potato beetlepopulations for up to 2 years after a single application of B. bassiana . Watt and Le Brun(1984) in USA demonstrated that soil application of B. bassiana successfully controlled ®rstand second generation Colorado potato beetle with 74 and 77% reduction in populations ,respectively. A granular formulation of M. anisopliae , BioGreenTM, has recently beendeveloped and registered in Australia against the soil inhabiting pest Adoryphorus couloniBurmeister (Jackson, 1999). It is reported that a single soil application of this product cansuppress the pest for 5- 10 years, considerably reducing the cost of control (Rath et al.,1995).
Pupal age susceptibility study showed that mortality of puparia decreased with increasingpupal age. On older puparia (96 h old) that had low level of infection, microscopicobservation revealed errant growth of hyphae without penetration (unpublished observa-tions). This may be attributed to the inability of germ tubes to penetrate the integument ofpuparia as it becomes hardened due to heavy sclerotization. Physical parameters such ascuticle hardness and its degree of sclerotization may aVect fungal germination and penetra-tion (St Leger, 1993). When compared with previous bioassay method, it would appear asif releasing pupariating larvae into treated sand resulted in higher puparia mortality thanwhen puparia were directly exposed to treated sand even at an early age of 12 h. Thisobservation suggests that a prophylactic application of mycoinsecticide before fruit set/infestation targeted at pupariating larvae would be the ideal strategy for control. Indeed,prophylacti c treatment with entomopathogeni c fungi has been found to be more eVectivethan curative application. Moorehouse et al. (1993) and Zimmermann (1994) reported thatgranules of M. anisopliae incorporated into potting media as a prophylacti c treatment toprotect glass-house-grown potted plants against Otiorhynchus sulcatus F. gave more eVectivecontrol of the pest than curative treatment. Prophylactic treatment of B. bassiana granulesused in sugar maple forest soils resulted in 18 months protection of the plant against pearthrips (Butt & Brownbridge, 1997).
Post emergence mycosis occurred in adults escaping infection as pupariating larvae in allthe experiments. Deferred mortality induced by entomopathogenic fungi has been reportedfrom various species of insects. Poprawski et al. (1985) showed that treatment of pupae ofthe onion maggot, Delia antiqua (Meigen) with entomopathogeni c fungi resulted in a highlevel of deferred mortality in adults that emerged from treated puparia. In Lepidopteraninsect pests, treatment of eggs with entomopathogeni c fungi can induce large deferredmortality in emerging larvae (Rodriguez-Rueda & Fargues, 1980; Maniania, 1991). Adultfruit ¯ies emerge from puparia by continuous pulsation of the pupal segments and wrigglingof the body and legs. Flies may have been contaminated either by contact with conidia on
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16 S. EKESI ET AL.
the pupal integument during emergence or by fungal penetration of the adult beforeemergence. Microscopic observations on adults that died within 24 h after emergencerevealed mycelial elements penetrating into the body of the ¯ies. Our 14 day limit ofrecording of post emergence mycosis in adults might have underestimated the overallmortality. The post emergence mycosis may play a signi®cant role in ®eld suppression. Thisalso suggests that these same isolates could be used in protein bait stations against adultfemales seeking protein to mature their eggs. The compatibility of these isolates withcommercially available baits/attractants, however, warrants further investigation .
We conclude that M. anisopliae ICIPE 18 and 20 are promising candidates for soilapplication against fruit ¯ies where the three tested species coexist. Although various bioticand abiotic factors such as inoculum density, micro¯ora, soil texture, temperature andmoisture can in¯uence inoculum threshold in the soil (McCoy et al., 1992), survival offungal pathogens in an edaphic habitat is reported to be enhanced relative to an epigeal one(Ferron et al., 1991) and many of them can survive saprophytically in soils, particularlythose rich in organic matter (Butt & Brownbridge, 1997). This has been attributed to thefact that infective propagules are protected from the extremes of temperature and ultravioletradiation. Several larval-pupa l parasitoids, however, attack all the three species of fruit ¯iestested in this study (White & Elson-Harris, 1992). The ideal fungal isolate should haveminimal to no eVect on the activities of other natural enemies within the agroecosystem tobe tested. This together with the eVect of environmental factors on the selected fungalisolates under diVerent soil types should be the subject of next investigation before initiating®eld control.
ACKNOWLEDGEMENTS
We are grateful to Drs E. Seyoum and N. K. Gikonyo for their comments on the manuscript.The research was supported by funds from IFAD.
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