a* r 2016 Anastrepha A. suspensa Diachasmimorpha long- Anastrepha and Larry W Duncan , followed by the native c The braconid wasp, 30% of another tephritid pest ( 4 ca (Ashmead), has established and is widely distributed in Loew) that entered baiting traps for adult annihilation . Future work will assess management strategies, using killed Caribfly at either larval or pupal stage. Pupae were Correspondence to: LW Duncan,University Institute of of Florida, Food CitrusStation Research and Road, Lake and Agricultural Alfred, FL Education Science, 33850, Centre, USA. E-mail: 700 lwduncan@ufl.edu Experiment Institute of Food and Agricultural Science, University ofand Florida, Education Citrus Centre, Research Lake Alfred, FL, USA Plant Protection Department,Zagazig, Egypt Faculty of Agriculture, ZagazigInstitute University, of FoodResearch and and Education Centre, Homestead, Agricultural FL, USA Science, University of Florida, Tropical 1 ∗ c a b − managed with aerial insecticides,cern in but places spraying where Caribfly-infested mayidential groves areas. be are Alternative adjacent of management to options, con- res- fruits such as in bagging trees, removalbaits of dropped or fruits, pheromone usingbecause pesticide-related lures of and much sticky labourhas and pads, equipment shown are required. that very Recentludens study expensive managed to escape. icaudata Florida following its introduction in 1972 as a parasitoid of Caribfly, Daniel Carrillo A. suspensa L.), L.), a,b Heterorhabditis bacteriophora Loew), and . 200 IJs ca . Psidium guajava Prunus persica in fields infested by Caribfly. A. suspensa Eriobotrya japonica with 1 mL of Anastrepha suspensa 1 L.), peach ( feltiae L.) and guava ( ; entomopathogenic ; resistant pupae; susceptible larvae; emerging adults; bioassays; A. suspensa Alston), tropical almond, loquat, Guava industries are affected by L.), loquat ( Fahiem E El-Borai, 1 a Loew (Tephritidae) A. suspensa Moreover, because larvae can be found in : 1220–1228 www.soci.org © 2016 Society of Chemical Industry 3 The feeding of larvae after hatching from eggs However, within the geographical distribution 73 2 1 , Terminalia catappa Eugenia uniflora 1 , the attack and the impact are more limited to 2017; Syzygium jambos Anastrepha suspensa Mangifera indica

and the exotic

A. suspensa Management of Caribfly is challenging because third-instar lar- vae leave decaying fruitssoils; dropped consequently, to both the larvae and ground pupaeand and are in pupate protected soils in in fruits from surface-applied insecticides. Adult flies can be Pest Manag Sci Several species of fruit flies (Diptera: Tephritidae) are invasivethat pests damage the quality of mature fruits infields commercial agricultural worldwide. 1 INTRODUCTION suspensa BACKGROUND: Caribbean fruit flyfrom (Caribfly) eggs is oviposited a into serious(EPN) fruits economic by isolates female to adults. pestfruit-to-soil-dwelling Caribfly This because stages study in of of assessed laboratory development the bioassays of virulence as larvae of twelve that a entomopathogenicRESULTS: hatch starting Inoculation of point toward evaluation of management strategies for the Abstract management of Caribbean fruit fly, William K Heve, entomopathogenic nematodes for Biological control potential of (wileyonlinelibrary.com) DOI 10.1002/ps.4447 Research Article Received: 11 July 2016 Revised: 19 September 2016 Accepted article published: 22 September 2016 Published online in Wiley Online Library: 14 Decembe Surinam cherry and guava. of rose apple ( L.), among others. tropical almond ( Surinam cherry ( laid inside fruits causes rots andfruit-damaging blemishes. Among flies the numerous is the Caribfly ( Caribfly in southern Florida, Jamaica, Hispaniola, CubaRico, and among Puerto others. more resistant to EPN infections than larvae.than Adult that emergence observed from in inoculated filter pupae paper in assays. soilpupae Longest microcosms in or was soils, largest significantly steinernematids whereas lower suppressed shorter emergence heterorhabditids ofwere were more caused more adult by infectious Caribfly exotic from nematodes to Caribfly larvae. The highest mortalities of infested fruits in commercial packages, Caribfly hasa been quarantine declared pest in all fly-free zones. which can infestas fruits mango of ( tropical and subtropical crops such indica CONCLUSION: Entomopathogenic nematodes reduced the developmentsuggesting of that Caribfly EPNs larvae have and potential pupae for to biological control adult of in our bioassays, biological control Keywords: © 2016 Society of Chemical Industry the virulent EPNs, in orchards infested by

1220 1221 ) 1 1 − − were C for 5 ∘ ,ofthe 26 , 90 25 . = i 1 − P in soil assays G. mellonella and were inoculated with 60 = C for 1–2 weeks before i ∘ P 2000 IJs mL were inoculated at initial IJ 0.5 ca A. suspensa ± ) of the appropriate EPN, using a 1 wileyonlinelibrary.com/journal/ps − All stock suspensions were diluted C until larvae pupated in treatments ∘ 24 A. suspensa , diameter 55 mm) in a small petri dish TM 8.1%. For controls, tap-water was applied. Five repli- 0, 25, 50, 100, 150, 200, 250, 300 and 400 IJs larva ca = i Three last-instar larvae of susceptible P 27 C. After 7 days following the date of inoculation, the number ∘ Ten third-instar larvae were placed on moistened soil and inocu- For treatment to pupae in soils, ten pupae were buried in grooves The same procedures were applied for nematode treatments selected isolates of EPNs (Table 1) and incubated at 27 2.2 Preparation ofLast-instar EPN inocula larvae of days. Cadavers were transferredof infective to juveniles White (IJs). traps for emergence (diameter 5 cm). The ten larvae wereIJs inoculated (equivalent with 1 to mL 200 of IJs 2000 larva pipette. 2.3 Virulence ofTen EPN species actively to moving Caribfly in third-instar filter paperfilter larvae assays papers were (Whatman placed on doubled more virulent EPNs at 60 and 90%Ten mortalities third-instar respectively larvae of densities also inoculated with 1 mL of 600 IJs (equivalent to 200 IJs larva A quantity of 22 g of autoclaved sandygravimetric soil moisture (moistened content) to was 2% added initial to a5 petri cm). dish The (diameter top of thebetween soil the lid was and levelled, the allowing soil enoughand for fly air emerging around. space adult fruit flies to move lated at 2000 IJs 1 mLmoisture of to tap-water, thereby adjusting the finalcates soil for each EPN27 treatment were made and thenof pupae incubated formed at was recordedments and for then adult maintained emergence. in the treat- (depths 2–5 mm, one for eachthe ), same inoculated way and as incubated the in larvae ininoculated soil pupae assays. were recorded Emerging between adult 2 flies and from 3the weeks following date of inoculation. Allrepeated twice. experiments in soil microcosms were 2.5 Infectious nematode densities, and served as acontrols positive were control treated for with nematode tap-water inocula.EPN that inocula. was Negative All used plates for were preparing of sealed using moisture. parafilm Five to replicates preventplates loss were were incubated made at for 27 eachbetween EPN 1 and isolate. 4 All that days could following not the pupate date butearly of died mortalities. inoculation. on Final Larvae filter (overall) paperadults mortalities were emerged were recorded from as confirmed pupae. after larvae Nematodes and pupae inside that could cadavers notmicroscope (killed eclose) to were confirm observed that under they the were killed by EPNs. to ten pupae perdue to petri EPN dish. treatment The wasnumber corrected proportion for of of natural pupae deaths pupal of inadults mortality a that controls large survived EPN by treatment subtractingthat from survived the the in proportion the proportion of controls. adults All of were experiments repeated in 2 filter times. paper assays 2.4 Virulence of EPN species to by adding enough water inin such bottles a way and to thenbeing avoid overcrowded kept used IJs at for 14.5 experiments.to separate The active Baermann IJs funnelsnematodes from were were the used dead immediately nematodes,were concentrated applied and and adjusted the after to active stock suspensions Stein- Fabricius), L.) in citrus Susceptible Steinernema 11 , 5.1] from Cen- Varying degrees 10 Bactrocera tryoni and ) using standard 9 = 3 Wiedemann), the Mexi- 30 cm Heterorhabditis bacterio- Boddie), corn rootworm , × 80% of fruit flies such as the Cylas formicarius . The species or isolates of ≥ 30 × Heterorhabditis Loew), the European cherry fruit However, EPNs can occur at very Diaprepes abbreviatus . 8 infect and kill damaging insect pests A. suspensa A. suspensa The objective of this study was to select (Rossi) and many more. Ceratitis capitata Helicoverpa zea 18 , 7 : 1220–1228 © 2016 Society of Chemical Industry 15 – L), the Queensland fruit fly ( 5 A. suspensa 73 Cincages(30 infected and killed root ( ∘ 75% suppression of the majority of ) in Third-instar larvae were selected for studies ≥ remains a pest in commercial guava groves, requir- Steinernema carpocapsae EPNs have not been used to manage Caribfly, per- Because EPN species vary in efficacy against specific 20 2017; , Anastrepha ludens Heterorhabditis 13 , 19 14 spp.), sweet potato ( . 12 S.feltiae The latter is more similar to field soil conditions. Ento- Bactroceraolae and 17 , 15 Diaprepes and 16 A. suspensa Rhagoletis cerasi Filter paper in petri dish assays as well as soil microcosms have Entomopathogenic nematodes (EPNs) of the genera Diabrotica ing other management options,soil-dwelling especially stages. those that target the haps because of limited information on virulenceA. of suspensa nematodes to phora Froggatt), promising nematode species forment field strategies trials for to develop manage- been used extensively to study the pathogenicity ofpests. EPNs to insect ernema in soils and are endemic in almostSome all agricultural of and natural these soils. nematodescentral and have southern been Florida. retrieved from groves in Pest Manag Sci Caribfly werereared at retrieved 26–28 from infested guava groves and 22.1 MATERIALS AND METHODS Rearing colonies of Can entomopathogenic nematodes contribute to Caribbean fruit fly IPM?but www.soci.org when they appeareddarkened mouthparts cream or well-developed to creeping welts.the pale Because majority yellowish of pupae andto produced had in dehydration, dry deep vermiculitesvae 1500–2000 died were owing transferred from healthy-lookingCandler larval sandy third-instar diets soils to lar- moist [7% autoclaved moisture fine (w/w); pH tral Florida and allowedstudies. to develop into 4–7-day-old pupae for mopathogenic activities of 12 isolates or species oftodes infective nema- were examined usingtheir both bioassays efficacy in to order the to pest establish targets, it is necessary to determinerange virulence for of a wide species or isolates of procedures. greenhouse and fields have beenof reported commercial products following of application EPNs fortrol augmentative of biological con- of success (i.e. groves, corn earworm ( ( mole crickets and scarabaeid beetlesinsect in turf pests and that lawns exit and other fruits and pupate in soils. low population density into soil achieve habitats, adequate requiring augmentation observed pest that management. Previous studies have fly ( Mediterranean fruit fly ( can fruit fly ( insect pests are notinfections. able to develop induced immunity to EPN EPNs that consistently performededly well showed in both high assays performancebe or the in repeat- more soil virulent assayslent EPNs EPNs were to realized deemed Caribfly. in Moreover, to comparing the the their LD first more values procedures viru- (or infectious werehave juvenile discriminated densities), been which by used in previousspecies among studies many. to identify the most virulent for assessment of their biologicalgroves. control potential for Caribfly in , H. , et al. spp., killed -group -group H. indica were more : 1220–1228 Sr-Brt Sc Sx-Kelly Sg-Na Srr Sf Sd-Brt Hi-Brt Hi-Homest. Hz-Brt Hf Hb-Kopp 73 S. feltiae H. floridensis S. glasseri S. glasseri Steinernema and 2017; S. glasseri and 22 22 22 22 22 22 22 22 22 22 and , , , , , , , , , , 23 21 21 21 21 21 21 21 21 21 21 (isolate from Homestead, FL), Pest Manag Sci S. diaprepesi S. carpocapsae , , were stronger than those of the genus S. carpocapsae larvae (results not shown) used as posi- m) References Symbol used H. indica , μ b22 IJ of nematode S. feltiae (isolate from Bartow, FL), , was used as the body length of 1000 > S. rarum , H. bacteriophora H. indica or both genera (Table 3). Shorter heterorhabditids G. mellonella S. glasseri , S. riobrave 0.05. Heterorhabditis , ≤ and (Homestead isolate), which was recently retrieved from guava P A. suspensa In filter paper assays, The significant sources of variations in overall (final) mortalities 70%) than efficacious (corrected overall mortalities in≥ repeated experiments (Figs 4A and 4B). In soil microcosms, the two isolates of H. floridensis replicates using the regressioncurves) lines and (mortality then compared versus amongspecies IJ using the density Tukey’s more multiple comparison virulent of nematode meanstest) (Tukey’s at HSD higher mortalities of Caribfly larvae into both IJ assays body were lengths unrelated (Table 3, Figs 4B and 4D). 3RESULTS 3.1 Early andObservations final mortalities in of third-instar both Caribfly larvae assays100% of were the similar. Alltive EPN control. inocula Infections killed of larvaeof by the EPNs majority caused early ofUninfected mortalities inoculated larvae developed Caribfly into viable normal larvae pupae from (Fig.adult which flies 1E emerged and later in Fig. treatmentsThe 2). (Figs highest 1H population and of 1J adults and thatCaribfly Fig. developed larvae 3). from were third-instar obtained in the85 controls and and ranged 98% between (Fig. 3). included IJ body length,individual which is EPN one species of(filter (Table the 1), paper distinct features the assays of experiments type or were repeated of soil and testing the microcosms),tode interactions bioassays species, between bioassays the and nema- repeating number experimentsmortalities (Table of 2). of Final times third-instar larvaebody were lengths negatively of correlated IJs. with ities However, the of relationships larvae between and mortal- the IJ genus body lengthsSteinernema of EPN species belongingachieved to higher overallerorhabditids mortalities (Table 3, of Figs 4A larvae and 4C). than Among longer het- zealandica H. bacteriophora H. indica S. diaprepesi ) i P were 0.1 in i www.soci.org WK Heve Overall ≥ P 29 , 28 -statistic was t © 2016 Society of Chemical Industry Natural area in Central FloridaExotic; isolate not knownExotic; isolate not knownCentral Florida (Bartow) 1130 Central Florida (Bartow)Florida; isolate 511 not known 849 1002 685 562 Exotic; isolate not known 622 Origin of isolate in Florida Body length ( 8 ) using a regression are coefficients and i were estimated over P h j 90 + = at which 60 and 90% final i ) and i P 90 P k = ( mortalities in controls when i h P ≈ and C: petri dishes (replicates at ∘ + . Five replicates for each 1130)/2], of published body lengths of 60 1 2 and − + ) = i i P 60 (Koppert isolate) Europe; exotic 588 P ( = i k P (isolate ALL) Exotic 558 [(1002 -intercept is the predicting variable) was established = y ≈ i y (Bartow isolate)(Homestead isolate) South Florida (Homestead) Central Florida (Bartow) 528 528 P m group Central Florida (Kelly) 0IJslarva μ = i 29 , ). Variables P 1 28 − is the nematode density, Species or isolates of EPNs tested against third-instar larvae and pupae of i P is the final mortality (not corrected because of the low a y 0IJslarva Mean value, 1066 Detailed information can be obtained in a report on the isolates, except = The relationship between final larval mortalities and initial in our statistical analyses in Table 3 because of the similarity in body lengths of IJs between these three nematode species. Steinernema carpocapsae Steinernema glasseri- Steinernema glasseri Steinernema rarum Steinernema feltiae Steinernema diaprepesi Heterorhabditis indica Heterorhabditis zealandica Heterorhabditis floridensis Heterorhabditis bacteriophora a Steinernema riobrave Isolate Table 1. groves infested by Caribfly in south-eastern Florida. b is a constant (or the i used to compareor observations between between bioassays. Having repeated confirmedof experiments the data normal distribution usinganalysis the of variance was normal performed to quantile–quantile determine the majorof sources plots, variations a in three-way correctedby overall deducting mortalities proportions ofthose larvae, of of obtained EPN final species treatments. mortalities Analysisto of in examine correlation was controls the used from between strength overall larval of mortality (corrected for association deadcontrols) larvae and (or IJ body lengths linear of the relationship) variousfor EPN both species sandy (in Table soil 1) and filter paper bioassays. nematode densities ( where mean values ofcontrols), dead third-instar larvae between 0 and 0.1 in wileyonlinelibrary.com/journal/ps (each pipetted in 1 mL of water) inmoisture the 8.1%). sandy soil Only microcosms (total tap-water was(or applied to controls) obtain at treatments 2.6 Data analysis R software (i386 v.3.2.2;for R the Core statistical Team, analysis. Vienna, Meansobserved Austria) variables and was were standard calculated, used errors and (SEs) the paired of all mortalities were assessed afterments adults were repeated emerged, twice. and the experi- (overall) larval mortalities were achieved, respectively. made and then incubated at 27 analysis for estimation of were numbered 1 to 5 so thatvidually their be corresponding fitted data could against indi- nematode density ( using a polynomial regressionEPN model treatment: for each replicate of every j P

1222 1223 per (only infected H. bacteriophora (only in experiment 1) (only in experiment 2) 0.05 not shown). In both < P S. glasseri wileyonlinelibrary.com/journal/ps , (only in experiment 1) and the longest H. bacteriophora S. diaprepesi S. riobrave group] in soil microcosms. Generally, pupal mortal- (isolate Hi-Brt) (only in experiment 2) (Figs 5A and ): (A) active third-instar larvae; (B) pupae; (C) filter paper assay; H. indica S. glasseri- A. suspensa filter paper assays, except and 5B). In thewere filter not paper different between assays, thetreatments proportions (results control for of and Tukey’s HSD the at emerging majority adults of EPN experiments, the lowest populations of emerging adults (between 15 and 30%) were observed in treatments of in experiment 2), steinernematids [ and ities due to EPN treatmentsand in 30%, filter whereas paper those assays in were the betweenand soil 60% 0 (Figs microcosms 5C were and between 5D). 10 H. (Figs 4C and 4D). 0.001 respectively. < P -group achieved the lowest S. glasseri 0.01 and and < P -test, *, ** and *** signify that observed variables between experiments 1 and 2 for each nematode isolate or species are S. glasseri t 0.05, and < isolate from Homestead. The nematodes isolate from Bartow was more efficacious in : 1220–1228 © 2016 Society of Chemical Industry P S. riobrave 73 , H. indica 2017; H. indica S. diaprepesi , S. rarum Observations in both bioassays following EPN treatments to Caribfly ( Early mortalities (not corrected for mortalities in controls) observed at the larval stage following inoculation of third-instar larvae in filter pa Pest Manag Sci more larvae (45–95%)killed when by compared with larvae (15–65%) Figure 2. However, the soils than the (D) soil microcosm; (E) early mortalitylarvae of infected developed larvae; into (F) pupae, mixture which ofnearly appeared infected normal, and dark but uninfected black; fewer pupae adults (H) formed emerged uninfected following from inoculation pupae soils of were than larvae; normal, in (G) brightly filter gold paper assays; to (J) brownish; adult (I) Caribfly. inoculated EPNs pupae were appeared found in lacerated E and G. assays. According to the paired Figure 1. significantly different at Can entomopathogenic nematodes contribute to Caribbean fruit fly IPM? www.soci.org 3.2 Emergencemortalities of adults from inoculated pupae and pupal In both assays, theadults maximum were observed population in densities the controls of80%. and emerging ranged Adults between that 70 and emergedments from in soils inoculated were pupae significantly fewer in than those EPN observed treat- in the zealandica final (overall) mortalities (1–30%) of larvae in soil microcosms. 7 8 6 6 16 − − − − 0.01, − et al. 10 10 10 10 < 10 × × × × P × -value P 2 < : 1220–1228 0.05, 73 < P in experiment 1, , thereby reducing 2017; 90 -test: *, **, *** and **** t = i P -value A. suspensa F Pest Manag Sci Significance level. achieved = -value P achieved by all the four nematode species in test; 90 F- = i were significantly different between experiments P H. bacteriohora D statistic for = value H. bacteriophora 1 and 2. Only experiment 2. 4 DISCUSSIONThe AND killing CONCLUSIONS ofsome large extent, proportions pupaepotential of has to demonstrated third-instar break the that larvae life EPN cycle and, of species to have compared with F- 60 60 H. = = i i and (Bar- P P or for values www.soci.org WK Heve 60 = i P S. feltiae © 2016 Society of Chemical Industry H. indica mean sum of squares; = and S. carpocapsae a ) for 60 was observed between = S. feltiae i P 60 = i . However, the difference in P A. suspensa sum of squares; MS in experiment 1 or 2 (Table 4). Simi- in experiment 1 between = 60 repeating experiments 10 0.776 0.0776 2.728 0.003662 = × i P SE) denote difference between total pupae recorded and pupae that developed to adult in each treatment at AB ± C S. feltiae was not significantly different from 0.0001 respectively. H. bacteriophora and repeating experiments 10 1.449 0.1449 5.095 1.32 bioassaysbioassays 10 1.403 0.1403 4.931 2.30 < × × × P repeating experiments 1 0.426 0.4257 14.967 0.000149 or Three-way analysis of variance for the sources of variations in final mortalities (corrected for dead larvae between 10 and 20% in controls) × Out of total pupae recorded, some pupae developed to adult Caribfly in treatments following inoculation of third-instar larvae: (A) and (B), (Bartow isolate) was not significant. In experiment 2, degree of freedom; SS = S. carpocapsae 0.001 and df S. feltiae a EPN species Bioassays BioassaysRepeating experimentsInteractions EPN species EPN species 1 1 0.951 0.736 0.9510 0.7362 33.436 25.881 2.87 8.47 of third-instar larvae of Caribfly ( Source of variationsEPN species (specific IJ body lengths in Table 1) 10 5.128 df 0.5128 SS 18.029 MS Table 2. Within groups (residuals) 196 5.575 0.0284 – – < tow isolate) was significant. The wileyonlinelibrary.com/journal/ps for values in experiment 2 between 3.3 Discriminationselected of the more virulent nematode isolates No significant difference in Figure 3. experiments 1 and 2 in filter paper assays respectively; (C) and (D), experiments 1 and 2 in soil microcosms respectively. Paired on top of paired bars ( bacteriophora larly, the difference in H. indica of P

1224 1225 spp., 0.789 0.043 0.048 0.009 34 , 33 e * * ** in controls) and Steinernema 087 238 0.262 591 51258 0.089 52 ...... 0 0 0 0 0 − − − − − spp. and A. suspensa Rapid respiration by actively 32 , 31 wileyonlinelibrary.com/journal/ps Heterorhabditis species Both genera SE) for each EPN isolate or species indicate difference Mortalities due to Steinernema ± 0.27 0.358 0.15 0.745 0 0.570.410.360.36 0.186 0.359 0.428 0.206 − − − − − − In this study, shorter heterorhabditids were more efficacious in compared with motionless and diapausing pupae which are noted for very low oxygen consumption. killing larvae than longer heterorhabditids. Perhaps, smaller nema- todes were more likely to penetrate smaller openings into these moving Caribfly larvae may produce more respirationwhich byproducts, may attract EPNs to kill more larvae than pupae. d 0.038 0.007 0.002 Cer- Bac- species ) between corrected final mortality of third-instar larvae and IJ body length of EPN R and 0.01 respectively. * ** ** 0.001 respectively. All analyses of correlation (Table 3) were performed for the linear relationship < < P 90 97 4084 0.252 28 0.643 83 0.080 ...... -test: *, ** and *** on top of paired bars ( RP RPRP P t 0 0 0 0 0 0 Mortalities due to Heterorhabditis − − − − − − 0.05 and 0.01 and -value for correlation ( < a P < P P = P Anastrepha fraterculus spp., respectively, tested in filter paper assays; (C) and (D), results for As shown here, larvae of Caribfly , 0.05, 30 , < 29 , P 27 Steinernema : 1220–1228 © 2016 Society of Chemical Industry Filter paper Soil microcosm 73 Anastrepha ludens spp. and 2017; , among others. Analysis of correlation for linear relationship between overall (or final) mortalities (corrected for dead larvae of Following inoculation of third-instar Caribfly larvae in repeated experiments: (A) and (B), results of corrected final mortalities for the AB Pearson correlation coefficient; CD b c = Pooled data from experiments 1 and 2 for the individual bioassays. R Controls were not included in the analysis of correlation because of the corrected larval mortalities following EPN treatments. Linear relationship was extremely weak. 12Filterpaper Both Soil microcosm Both e a b,c d Experiment1Filterpaper Bioassay Table 3. 2 Soil microcosm IJ body length of EPN species in bioassays species; * and ** indicate significance at respectively, tested in sandy soilbetween microcosms. experiments 1 Paired and 2 at between body length of IJs (independent variable) and corrected final mortalities (response) in EPN treatments, excluding data in controls. Pest Manag Sci populations of adultCaribfly flies. suggests The that at incidenceObservations least of in Caribfly this EPNs larvae study were are inside similar hosts to dead those to reported EPNs. on Figure 4. Heterorhabditis atitis capitata trocera oleae Can entomopathogenic nematodes contribute to Caribbean fruit fly IPM? www.soci.org are the most susceptibletodes soil-inhabiting stage because to Caribfly virulent larvae nema- are very active and highly motile ired 90) c – et al. = 0.199 is the Higher (pi i P P -tests 14 t : 1220–1228 73 ** ** 60) ,where j 0.001 respectively. = 212002 – 381 – 009 + . . . . < (pi ) i 2017; P P Nematode-repelling P group) in soil bioas- ( 9 h + 2 ) i SE) P 0.01 and ( ± < k P S. glasseri- = 8.3 b 0 8.3 a 0 36.7 b 0 24.3 b 0 y Pest Manag Sci ) ± ± ± ± a 1 (mean − 0.05, and < 90 P = i P S. glasseri , SE) However, IJs may have simply infected and killed ± 35 Estimates (IJs larva in experiment 2 Paired 7.9 a 142.4 5.6 ab 178 7.1 ab 210 12.1 b 212 ± ± ± ± (mean S. diapresi 60 = 0.05. i P ≤ P respectively, between experiments 1 and 2; ** indicates significance at es (0–10%) of death rates of larvae in controls. Different letters against openings (e.g. spiracles, mouth and anus)tization owing and largely thickening to of sclero- the cuticlecause into of puparial pupal cells resistance is to a EPN major infections. says could be attributed to the fact that their IJs have more fat more Caribfly adults in soils than in filter paper assays. chemical exudates from pupating larvae may alsoing be released pupation. dur- pupal mortalities achieved by thematids largest ( and longest steinerne- b 90, = i SE) P ) ), 9 66.4 90% according to the polynomial function ± < ± and ) 1 www.soci.org WK Heve 60 − (mean = i P 90 A. ludens = C. capitata i P © 2016 Society of Chemical Industry -tests for t SE) obtained from lines fitted for each replicate for every EPN isolate ± 9 c n.a 55.6 2 a 160.8 18 bc n.a 81.2 16 ab n.a 32.4 in experiment 1 Estimates (IJs larva 90 = ± ± ± ± i SE) indicate significant differences between the two bioassays at P ± (mean 60 and = i 60 P -values of paired = P i P Reports explained that the closure of all 30 ) and Mediterranean fruit fly ( , represent 29 , 90) 27 , = 9 (Hb-Koppert) 24.4 SE) indicate significant differences according to Tukey’s HSD at (pi B. olae (Sc) 140.8 P ± The mean values of and (Hi-Brt) 109.2 (Sf) 67.2 Observations following inoculation of pupae in the bioassays: (A) and (B), are experiments 1 and 2, respectively, for results of surviving adults not applicable because optimum final larval mortalities were 60) = = 0.01. (pi < n.a Mortalities in EPN treatments were not corrected because of low mean valu P CD AB S. carpocapsae S. feltiae H. indica values (mean IJ density. P b c a EPN isolates H. bacteriophora Table 4. tests: *, ** and *** on top of paired bars ( olive fruit flyamong ( others in previousable studies, suggesting to that EPNs penetratepupal are resistance. and not infect pupae of fruit flies because of wileyonlinelibrary.com/journal/ps small Caribfly larvae. Onsteinernematids the other were hand, more theadults longest effective (or following at largest) inoculation of suppressingmortalities pupae emerging due in to soil EPN microcosms.and species Pupal in similar treatments to were generally those low reported on Mexican fruit fly ( t- Figure 5. that emerged from inoculated pupae; (C) and (D), experiments 1 and 2, respectively, for corrected pupal mortalities due to EPN species or isolates. Pa

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Diachasmimorpha longicaudata on 2 Sarwar M, Quarantine treatments for mortality of eggs and larvae of 1 Weems HV, Jr, and Heppner JB, Caribbean fruit fly ( 3 Mossler MA and Crane J, 4 Perea-Castellanos C, Pérez-Staples D, Liedo P and Díaz-Fleischer F, 9 Grewal PS, Ehlers RU and Shapiro-Ilan DI, 8 Campos-Herrera R, Johnson EG, EL-Borai FE, Stuart RJ, Graham JH and 5 Baranowski RM, Wasps6 sting flies, Meirelles RN, 60–40. Redaelli Research LR and ’87, Ourique UF/IFAS, CB, Comparative biology of 7ThompsonCR, of Agriculture andFlorida, Prof. Consumer Lukasz Stelinski, Services JoshCREC-UF/IFAS (FDACS) in Q Lake Fluty Alfred, in Florida, and and Dr all Gainesville, persons Nabilwe whose could Killiny names not of mention. 17 Koppenhöfer AM and Fuzy EM, Effect of soil type on infectivity 18 Girón-Pablo S, Ruiz-Vega J, Pérez-Pacheco R, Sánchez-García JA and 19 Burditt AK, Jr, Lopez DF, Steiner LF and von Windeguth DL, Application REFERENCES 16 Shapiro DI and McCoy CW, Virulence of entomopathogenic nematodes 20 Bronson CH and Gaskalla R, Procedures Manual for Mass Rearing the 10 Lacey LA and Georgis R, Entomopathogenic nematodes for control of 11 Miles C, Blethen C, Gaugler R, Shapiro-Ilan12 D and Peña JM, Murray Carrillo T, MA and Using Hallem EA, Variation in the susceptibility13 of Poinar GO and Grewal PS, History of entomopathogenic nematology. 14 Dolinski C, Entomopathogenic nematodes15 against the Shahidi main NS, guava Ansari MA and Moens M, Diversity in pathogenicity of 47 H. – and 41 , S. rio- Rossi) 11 , (Bartow (Sc) and H. indica H. indica H. indica values at 10 However, were more 60 40 = – and i indica 38 , P 38 are widely dis- . 30 , were more viru- H Bactrocera zonata – S. carpocapsae (L.) in Florida citrus S. feltiae 27 Bactrocera olae Wiedemann), Mexican S. carpocapsae L.), Queensland fruit fly , suggesting similarities in 49 60 (Bartow isolate) and the three S. carpocapsae = has not been noted for being i is more entomopathogenic to P H. bacteriophora achieved smaller H. bacteriophora (Hb-Kopp), and H. indica and (Bartow isolate) or 37 Loew), peach fruit fly ( on the one hand and Rhagoletis cerasi S. feltiae H. indica Ceratitis capitata Diaprepes abbreviatus S. fetiae : 1220–1228 © 2016 Society of Chemical Industry in the same way that the non-native will be better adapted to the climate and has S. feltiae and 73 S. fetiae Froggatt), olive fruit fly ( S. feltiae H. indica has been reported as the most virulent EPN to to be the most infectious nematode to larvae of 14 , H. bacteriophora and 36 2017; , 11 , Similarly, our tests by filter paper bioassays did not H. indica 14 9 Nevertheless, future work will include the native 27 (Sf) to be the more virulent EPNs to Caribfly, based on A. suspensa (Bartow isolate) on the other. However, overall mortalities . Anastrepha ludens 48 S. feltiae is used to manage We conclude that EPNs have potential for Caribfly IPM. Two H. bacteriophora We considered the native Bactrocera tryoni extremely virulent to a wide range of fruit flies. isolate). Until this study, their efficiencies observed inbioassays. the In previous prescreening studies, tests testsreveal using by filter both paper assays didC. not capita S. feltiae and extensively applied to many target insect pests. tributed in temperate, subtropical,semi-arid regions; Mediterranean, their tropical commercial products and United are States, available Europe, in etc., the and have been preferred to show that either virulent to Caribfly larvae. However, the twowise species to proved Caribfly other- in soil assays,ter supporting paper assays previous are reports artificial and that some fil- infective nematodesable are to not achieve their fullcompared biological with potential soil in assays. filter paper assays (Bartow isolate) to assess themanagement EPN strategy species in richness groves approach infested asindigenous by a Caribfly, because the similar virulence to the exotic isolates. lent to Caribfly compared with the indigenous non-native isolates, exotic species Pest Manag Sci The first author thanks the Department oftology, Entomology and University Nema- of Florida,Assistantship for that awarding paved himopportunities the a into biological PhD way control Research forappreciate and pest contributions, the management. recommendations study We andport and of technical research Vanessa sup- SimoesNematology Department, Dias University De of Florida, Castro DrUSDA Nancy of in Epsky Miami, the of Florida, Suzanne Entomology Frazer of and the Florida Department ACKNOWLEDGEMENTS Southern Florida has subtropicalin to which tropical products climatic ofmanage conditions these exotic isolates may prove useful to Can entomopathogenic nematodes contribute to Caribbean fruit fly IPM?deposits in their body and areof likely time to and survive for still a remainemerge longer actively period from infectious pupae in (buried soilemergence. in until soils) adult to flies tunnel through soils for www.soci.org European cherry fruit fly ( ( and others. 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