13 2018 10 × production ) or per mg ). Groups of at 24 h post 11 90 14 OB/container) 6 c 10 13 in vivo × –LC 14,21 10 which together with 50 × 17–20 OB/larva). No significant to 5.97 11 11 occlusion bodies (OB)/larva 40 ha of banana crops using 10 10 11 ∼ × × 10 × Delia Muñoz b ) instars. Inoculation of L 5 larvae (1.00 6 Each baculovirus requires a specific combination )orfifth(L 14,22–24 4 Correspondence to: P Caballero, Dpto. Producción Agraria,de Universidad Navarra, Campus Pública Arrosadía s/n, 31006, Pamplona,E-mail: Navarra, Spain. [email protected] Instituto de Agrobiotecnología, CSIC-Gobierno de Navarra, Mutilva Baja, Spain Instituto de Ecología AC, Xalapa, Mexico Dpto. Producción Agraria, Universidad Pública de Navarra, Pamplona, Spain At present, the only viable option for large-scale production ∗ c a b virus production; biological insecticide; cannibalism technical constraints. For instance, cell culture production canto lead a variety of mutations inthe baculovirus biological genomes activity that can of reduce viruses, of baculoviruses involves usingand the so all host the insect baculoviruspests as products worldwide are used a currently against biofactory, producederent diff systems. using insect the high cost of cell culture media,of has suitable hindered reactor-based the production development systems. of conditions touloviruses maximize present its a narrow production. hostrange range, Although and others can most have be a produced bac- in wider several host host species. Trevor Williams, 4 in in vivo a system OB/larva) than recently molted L and to horti- face important in vitro 11 3–5 16 10 × (Esper) (: a,c* ), in larvae inoculated with a range of inoculum concentrations (LC This pest causes serious 9 on banana crops. Commercialization of this virus as a bioinsecticide requires an efficient In addition, 10 1–4 × Oihane Simón, 14,15 ; Alphabaculovirus; ChchNPV; ). The expenses associated with a )wasthemostsuitableforvirusproduction,producing1.80 6 to 2.11 11 in vitro 10 :1586–1592 www.soci.org ©2018SocietyofChemicalIndustry chalcites × The pathogenicity and virulence of this strain 74 4,7–9 Chrysodeixis chalcites 2018; larvae reared at a density of 150 larvae/container produced a greater total number of OBs (8.07 Chrysodeixis chalcites 6 10–13

) or in cell culture ( Commercial production of baculovirus insecticides requires an efficient and economicallytion viable system. occlusion As body obligatefor (OB) pathogens, replication, produc- viruses and require thisvivo living can hosts be achieved only in host larvae ( Pest Manag Sci opolyhedrovirus from southern Tenerife (CanaryChchNPV-TF1, was Islands), a named highlyectiveff e biological insecticidetrol for of con- this pest. Previous studies revealed that an isolate of C. chalcites nucle- 1 INTRODUCTIONThe looper, Keywords: the from a single container. ©2018SocietyofChemicalIndustry OB/container). CONCLUSION: The processes described here allowcient effi production ofcientffi su OBs to treat production system. RESULTS: The sixth instar (L Abstract BACKGROUND: A Chrysodeixis chalcites nucleopolyhedrovirus from the Canary Islandsfor (ChchNPV-TF1) has proved control to be of ective eff and Primitivo Caballero homologous host Alexandra Bernal, nucleopolyhedrovirus (ChchNPV) isolate in its Remarkably efficient production of a highly insecticidal Chrysodeixis chalcites (wileyonlinelibrary.com) DOI 10.1002/ps.4846 Research Article Received: 23 October 2017 Revised: 21 December 2017 Accepted article published: 3 January 2018 Published online in Wiley Online Library: 23 February systems, vaccine development and gene therapy, techniques, currently used in recombinant baculovirus expression uloviruses currently commercialized asproducts. the basis for insecticidal is comparable with that of the most pathogenic and virulent bac- cultural greenhouse crops in Spain and other parts of . erencesff di were recorded in mean time to death (165–175 h) or OB production per larva (3.75 damage to banana crops in the Canary Islands, larval weight (1.30 both these systems caninsecticides make at it competitive difficult costs. to produce virus-based ) is considered a major lepidopteranof pest Europe, in many parts and the Middle East. and showed a lower prevalence of cannibalism (5.4%) than fourth (L than lower densities (25, 50 and 100 OB/container), and a similar number to containers with 200 inoculated larvae (8.43 infected L molting produced six times more OB (5.72

1586 1587 7 10 × OB/ml, 8 10 ,5.57 8 × 10 8.5 cm (height) × × using the droplet 8 ,1.41 stages were inoculated 8 6 10 × concentration of OBs using 8 h prior to death), and incu- and L ), were inoculated with a range ∼ 90 5 6 stage: (1) recently molted (1–8 h 10.5 cm (width) ,L 6 4 ,3.56 Each group of larvae was placed in × 8 wileyonlinelibrary.com/journal/ps 8 10 × 8 h prior to death), larvae were weighed C until required for OB titration. For this, larvae were too small to be weighed with ∘ 8h priortodeath),atwhichtimetheywere ∼ 4 ∼ 20 insects were inoculated with 9.02 1 cm was placed in each container on top of a − OB/ml, previously calculated to kill between 90% 6 × 7 5 10 × × ). Inoculated larvae were placed individually in 30 ml plastic Larvae were checked every 8 h until death. When observed Toestimatecannibalism,theL 6 to be moribundindividually, and ( placed individually in microcentrifuge tubes until death. However, L and covered with a ventilated plastic lid.diet A piece of of semi-synthetic 10 cups with artificial dietMortality and was recorded incubated at until 8-hof death 25 intervals larvae to or of estimate pupation. each MTD.dye physiological Groups and age were sucrose inoculated solution withexperiment without food OBs was as performed negativebatches on controls. of The three insects. Larvae occasionswere were using weighed moribunderent ff di individually ( when they of 2.5-fold dilutions: 9.02 and 2.20 recorded daily. The experiment was performedusing onerent diff nine batches occasions of insects. 2.3ectff E ofGroups inoculation time of on OB 25 production L and MTD each with their correspondingthe droplet LC feeding technique. Larvae thatin ingested the inoculum 10 min werecontainers of placed 13.5 cm in (length) groups of 50 in rectangularplastic plastic grid. Groups ofwith 50 food dye larvae and sucrose of solution asof each negative larvae controls. instar Numbers that were reached inoculated were the partially pupal stage, or cannibalized totally larvae consumed), (that and virus-killed larvae were consistent precision. Therefore, we couldOBs/mg not obtain larval the weight value of larvae in (at this least 40 instar. for Afterand each larval death, stored stage all and at replicate) virus-killed were frozen post molting) and (2)L 24 h after molting had occurred (24-h-old and 50% of larvae in a sample. placed individually inlarvae microcentrifuge were tubes. homogenized in Followingfiltered 0.1% through death, SDS, muslin OBs and duplicate wereNeubauer samples extracted counting were chamber. and counted in a 2.4ectff E production of inoculum concentration on MTD and OB Five groups of 25 larvae (24-h-old L one of the rectangularpreviously. plastic As containers controls, with identical diet,lated as numbers with described of food larvae dye and weremine sucrose inocu- the solution, influence without OBs. ofval To inoculum deter- mortality concentration was recorded on atwhen the 8-h they intervals. MTD, were visibly Larvae lar- moribund were ( bated weighed individually in microcentrifugeupon OBs tubes were until extracted death, andwere duplicate counted where- using samples a from counting chamber. each The experiment larva formed was on per- six occasions usingerentff di batches of insects. feeding method. Larvae werephysiological inoculated times at during the one L of twoerent ff di which was expected to result in 90% mortality, cadavers were thawed, homogenized in 0.1% SDS, filtered through muslin and duplicate samples of OBs forin each a insect were Neubauer counted chamber. The influenceto of death instar (MTD) on the was mean analyzedintervals. time by recording larval mortality at 8-h , 8 6 5% 10 ± × larvae ), were 6 90% larval C. chalcites ∼ 26 and L , respectively. Cand70 5 6 ∘ C. chalcites 1 ,L 4 ± and L 5 ,L 4 Inoculated larvae were placed production systems also require 28 OB/ml for L 5% humidity, and a photoperiod of 16: 8 in vivo ± were obtained from a laboratory culture 10 C. All experimental procedures described in :1586–1592 ©2018SocietyofChemicalIndustry ∘ × 74 In general, when cannibalism is not an issue of ChchNPV-TF1 was characterized and selected C, 70 ∘ 23 Larval rearing density during the period that fol- 7 )wasusedtoinoculateeachinstar:5.56 1 2018; 90 ± larvae. OBs from virus-killed larvae were extracted, 24,25 and 9.02 C. chalcites NaCl and finally resuspended in distilled water. Purified 7 M Adults were fed 30% w/v diluted honey. The ChchNPV-TF1 10 × 27 In this study our aim was to evaluate the influence of inoculum In addition,cient ffi e mortality (LC 2.2ectff E and of mean larval time instar to on death OB production, cannibalism Groups of 50 recently molted (2–8of h each previously) of the fourth, fifth and sixth instars (L humidity. strain was originally isolated froma a natural single epizootic larva in collected aIslands during banana crop (Spain). in southern Tenerife, Canary infected insects. lows inoculation is also influential in the growth and survival of be an issue when rearingcan late greatly reduce instar survival larvae and overall gregariously, yields and of this OBs. concern, larvae tend to be reared attoreducehandlingtimes,whilekeepinglarvalstresstoaminimum the highest densities possible toavoidreductionsinhostweightgain.However,cannibalismmay insecticides for control ofcrops. this pest in banana and horticultural on the quantities oflarvae ChchNPV-TF1 from OBs the produced sameprove in valuable geographical for region. the commercial Our production findings of ChchNPV-based should concentration, growth stage at inoculation and rearing density mize the trade-offbetweenproduction. speed of kill, larval biomass and OB optimization of diverse biological variables, particularlystage the at larval inoculationinfluence and the inoculum total concentration yieldbe that of strongly precisely OBs. These determined two for parameters each need to virus–host system to maxi- occasions usingerentff di batches of insects. Pest Manag Sci As controls, identicalfood numbers dye and of sucrose solution larvae (10%Blue sucrose were dye), and 0.001% without inoculated Fluorella OBs. with This procedure was performed on three covered with a ventilateddied or plastic pupated. cap A and single concentration incubated producing until they individually in 30 ml plastic cups containing semi-synthetic diet, et al. odically with insects collected fromreared the same at island. 25 Larvae were 8 h (light: dark), on a semi-synthetic diet described by Greene established in 2007 at the Universidadusing Pública de insects Navarra (Spain), collectedife, from and banana maintained crops atAgrarias in the (ICIA), Tenerife Instituto southern (Spain). Canario Tener- This de colony Investigaciones was refreshed peri- 22.1 EXPERIMENTAL METHODS Insect source,Larvae rearing and of virus strain ProductionofahighlyinsecticidalChchNPVisolate www.soci.org the following sections were performed at 25 OBs were stored at 4 C. chalcites filtered through muslin, washedwith twice 0.1 with 0.1% SDS and once from among othersured strains by concentration–mortality due metrics, to andChchNPV-TF1 rapid its OBs speed were of high amplified kill. in pathogenicity, a single mea- passage in fifth instar starved for 12 hthe and droplet inoculated feeding with method. ChchNPV-TF1 OBs using 5.00 6 ± = 35 was ± et al. 6 C. chal- 0.001). 6 = 3.156; d.f. P instars inoc- :1586–1592 = 6 3.573; d.f. t ,andL 74 5 = t ,L 0.981, and L 4 5 = 2018; r ,L 4 larvae. L 6 0.001), with average ( 36 mg, respectively. How- < ± P 5; C. chalcites of OBs. Pest Manag Sci = 90 insects weighed 1.4 times more than 0.476), or the MTD of infected larvae. 41 and 230 6 = ± 13.8; d.f. P = t ( 6 0.905, = larvae and 176 h for 24-h-old L 48 mg and at between 165 and 175 h post infection, 6 0.05). B). Percentages of larvae reaching the pupal stage, larval 4,25 0.025) (Fig. 2A). Similarly, the efficiency of OB produc- ± A) Mean OB production of ChchNPV-TF1 in L F < = P 0.016) (Fig. 2B). Mean OB production per larva was posi- P larvae. Values above bars indicate average OB production.erent Diff = 5; P ever, this increase in larvalin weight the resulted average in number a of= 5.7-fold OBs increase produced per larva ( 168 h for L tively correlated with larval weight (Pearson’s Finally, MTD values did noterff di significantly, with a MTD value of cites letters accompanying values indicate significanterences diff between them (Tukey, cannibalism and virus-killed larvae in SE) live weights of 315 tion (OB/mg larval weight) increased by 4.2-fold in 24-h-old L ulated with their corresponding LC newly molted L Diseased insects died with mean bodyand weights between 358 290 Figure 1. prevalence of cannibalism than the other instars, and so L larvae compared with5 recently molted insects ( 3.3ectff E production of inoculum concentration on MTD and OB Virus-induced mortality was notInoculum registered in concentration the control didweight group. ( not significantlyectff a the final selected as the most appropriatestudies. stage for use in the subsequent 3.2ectff E ofNo inoculation time virus-induced on OB mortality production was andAt MTD registered 24 in h the after control molting, group. L 1, 10 5.3, ± 10 = 0.982, × www.soci.org A Bernal Finally, = ) prior to 2,24 29 r x F ( 1.03 √ 4 ± © 2018 Society of Chemical Industry 11 10 × (18%) and interme- ) were orally inocu- 6 SE values of 140 than in L 4 6 ± larvae took longest to die, 6 to 1.80 0.001), which increased from 4 < P 0.109), or pupated (2.8–8.4%), OB/ml) using the droplet-feeding = 8 stagesered diff significantly in MTD P 0.001) (Fig. 1B). Larvae that were not 6 10 larvae (24-h-old L 187.4, C until required for titration, whereupon < × (5.3%), highest in L ∘ 0.001), with MTD = P 2.4, 6 OB/larva in L < 20 2,6 and L = 8 P F − 5 (9.02 10 13.7, 2,24 1 h, respectively. ,L 90 F × 4 = ± C. chalcites (Fig. 1A). Production was positively correlated with 145.90, 2,24 6 4.23 F = ( ± 5 9 2,6 F 10 1 and 167 × ± 0.001). The L 0.012) (Fig. 1B). The prevalence of cannibalism, defined as larvae that disap- According to these results, although L < = -test. The correlation between OB production and larval weight victims of cannibalism either succumbed(79–86%), to polyhedrosis disease the prevalenceamong of instars which did ( noter diff significantly peared or wereinstar, being partially lowest in devoured, L eredff di significantly with 152 8.87 wileyonlinelibrary.com/journal/ps instar at inoculation ( OB/larva in L 3.1ectff E and of MTD larval instar on OB production, cannibalism No virus-induced mortalityThe was yield recorded in of the OBs control from group. each larva was significantlyectedff a by 3 RESULTS and OB production Abatchof600 2.5ectff E of larval rearing density on cannibalism rates lated with the LC which was significantly more prevalent in L larval weight across theP three instars tested (Pearson’s tions were determined by fitting Weibull survival modelsGeneralized using Linear the Interactive Modeling (GLIM) program. t was examined by Pearson’serentff di cient.ffi coe larval stages, MTD inoculation values times and recorded inoculum concentra- at ANOVA. Comparisons of larval weightsferent and ages OB (following production molting at and dif- 24 h later) were performed by larvae were stored at tainer was checked dailybers for of 8 pupae days and postdevoured) cannibalized inoculation. were larvae The noted (disappeared num- and orweighed immediately diseased partially prior larvae to death. were Following collected death, and virus-killed in the rectangular plastic containersviously. with Identical diet, numbers of as larvae mentioned were pre- inoculatedand with food sucrose dye solution, without OBs, as negative controls. Each con- method and distributed in groups of 1, 25, 50, 100, 150 and 200 diate in L this instar yielded greater numbers of OBs and showed a lower P values ( mally distributed. OB production values acrosssities larval were normalized rearing by den- square root transformation ( The means of final larvalval weights, weight OB values production of and each replicate OBs/mgto were lar- univariate calculated and analysis subjected of variance (ANOVA), when data were nor- batches of insects. 2.6 Statistical analysis they were thawed, homogenizedmuslin and in duplicate samples 0.1% of OBs SDS,chamber. were The filtered counted experiment in was through a performed Neubauer 10 times usingerent diff Softonic, Barcelona, Spain). larvae in each replicate were all normally distributed andjected were to sub- ANOVA and Tukey test.vival analysis, All were performed analyses, using except SPSS v. Weibull 23 sur- (IBM SPSS Statistics, the average prevalence of pupation,vae cannibalized that larvae disappeared (i.e., or lar- were partially devoured) and virus-killed

1588 1589 13 to and 10 8 × 10 12 × C. chalcites OBs/ml). B) 10 1.87 6 8 × ± 1.97 10 13 0.229) (Fig. 3B). × ± 8 = 1.33 10 0.001) (Fig. 3C). Of P 0.05). 10 ± × (9.02 < > × 12 90 P P 10 1.427; × = trast, individual larvae reared larvae in rectangular plastic con- 27.216; 5,54 F = wileyonlinelibrary.com/journal/ps 5,54 OB/container, respectively, whereas F 13 C. chalcites OBs, respectively), and the 100 larvae 10 6 OB/mg larval weight but did noter diff sig- × 12 8 10 10 1.86 × × ± 13 2.74 3.87 ± 10 ± × 13 9 A) Percentages of larvae reaching the pupal stage, larval canni- 10 10 × × Mean OB production varied from 7.19 larvae were inoculated with theMean ChchNPV-TF1 LC OB production per mg ofper larva container (OBs/mg) and in C) 24-h-old total L OB production tainers at larval densities:indicate 1, mean 25, OB production. 50, Same 100, lettersno accompanying 150 significant valueserences diff and indicate between 200. them (Tukey, Values above bars and 8.07 Figure 3. balism and virus-killed larvae rearedand at 200 larval in densities: rectangular plastic 1, containers. 25, Twenty-four 50, h 100, old 150 L nificantly among larval densities ( treatment was intermediate. By con However, the total OBcantly with production rearing per density container ( varied signifi- the densityproductive of 25 treatments or (with 50 5.72 larvae/container were the least 1.74 the gregarious rearing treatments, the containerslarvae with 200 were or 150 the most productive, with 8.43 1.10 8 11 19, 10 10 ± × × 0.425). = 6.66 P 1.19 ± had occurred 1% at 25 lar- ± 9 14, 295 6 ± 11 10 ± 10 × 1.003; × = OB/ml) resulted in 0.801). However, the 8 OBs per group of 25 5,54 = F 30, 318 10 to 2.11 P SE) larval weight imme- 12 1% in the 200 larvae den- 8 ± × ± 10 ± 10 × × 0.408, 0.962). By contrast, cannibalism = = 23, 354 4.63 ) or 24 h after molt to L P 6 ± ± 4,25 2%, and did noter diff significantly 9 F OB/ml. Also, OB production/mg larval ± 10 11 0.150; × 1% at the density of 200 larvae/container = 10 :1586–1592 ©2018SocietyofChemicalIndustry ± 21 mg, respectively) ( 0.022). × 74 4,40 0.835), ranging from 3.75 0.05). ± = 0.001) (Fig. 3A). By contrast, the prevalence of F < P = < 1.79 P 2% and 12 P ,newlymolted(L P 2018; ± 6 ± L 11 3.22; 10 A) Mean OB production per larva of ChchNPV-TF1 (OBs/larva) = × 13.69; 0.359, 13 and 327 4,40 = = 2% in the 25 larvae density to 75 24). Values above bars indicate mean OB production.erent Diff letters F ± + ± C. chalcites 4,40 4,25 6 F F 303 among densities ( between 11 The prevalence of larvae that reached the pupal stage varied (L sity ( virus deaths decreased85 significantly with rearing density from Pest Manag Sci lum concentrations tested ( to 5.97 OB/mg larva but did noter diff significantly among the inocu- depending on inoculum concentration.erencesdiff Similarly, were no observed significant ( in mean OB production per larva Figure 2. and B) mean OBin production per mg of larva of ChchNPV-TF1 (OBs/mg) ProductionofahighlyinsecticidalChchNPVisolate www.soci.org diately prior to death (317 3.4ectff E ofNo larval density virus-induced on OB mortality production The was dierentff recorded larval in densities thedid tested not control (1, significantly group. 25, influence 50, mean 100, ( 150 and 200) inoculated larvae). highest inoculum concentration (9.08 accompanying values indicate significantueserences diff (Tukeyt-test, between these val- ( increased significantly with rearing density from 2.4 vae/container to 14 weight varied from 1.30 the highest percentage of virus-killedthe larvae (97%) highest and overall therefore OB yield (7.17 7 6 39 C. , 10 10 20,26 et al. × × A. gem- Anticarsia The larval –AcMNPV –HearNPV C. chalcites Spodoptera :1586–1592 46 48 74 –HearNPV. S. frugiperda 138-fold higher 35,42,44 However, because 47 2018; and –SpliNPV, H. armigera 37 OBs/larva in nucleopoly- OB/larva the production Trichoplusia ni OB/mg larval weight or 14,25,50 9 11 9 OB/mg), 10 –SeMNPV, 7 10 10 × –SeMNPV system (1.31 × × 100-fold higher OB yield than 10 S. exigua Pest Manag Sci × ∼ 129-fold higher than the However, we were unable to calcu- 49 , Helicoverpa armigera and 5 52 At 5.7 30 9 The high OB yields suggest a remark- 51 S. exigua 10 and 14 Spodoptera littoralis × 47 45 larvae reared at high densities developed OB/mg), Spodoptera exigua H. armigera 7 OB/mg). 6 10 10 × AgMNPV OB/mg or 1 × or 240-fold higher than the 6 OB/larva, the productivity of ChchNPV-TF1 in tended to be lower than reported for other lepidopteran –SpexMNPV, 35 10 11 –AgMNPV system (1.38 × 10 × The productivity ratio (PR) may be a more valuable parameter The OB production process developed here resulted in remark- Rearing densityects eff on larval development and body weight growth stage withhighest similar inoculum yields concentration resulted of intion OB/larva. the of virus-killed greatest Nonetheless, larvae propor- the andproduced the greatest from overall each number of batchare OBs of consistent inoculated with insects.systems, those These such of results as other baculovirus OB production the highest density (200 larvae/container) was similar to theyield overall observed at the density ofdensity 150 larvae/container. of Because the 150 larvae/containerfewer requires larvae the to inoculation set oflarvae/container 25% up treatment, each the density container,was compared of selected with 150 as the larvae/container density. the 200 most appropriate post-inoculation rearing able yields, which as farobserved as for we are any aware,tem lepidopteran are among nucleopolyhedrovirus reported the pathosys- highest to date. With 1.81 was higher than the productivityhedroviruses reported in for permissive other hosts nucleopoly- and 6 which range between 3 OB/mg), Each of these viruses was used to inoculatespecies their homologous and host the highestquantities production of of inoculum OBs closetions. was to obtained the 90 using or 95% lethal concentra- have been examined in some species ofMamestra Lepidoptera. For example, brassicae faster, had adisease smaller than body conspecifics size reared and at lower were densities. more susceptible to so that gregarious rearingproduction was system. feasible However, in asincreased the with the ChchNPV-TF1 increasing prevalence OB rearing of density, the cannibalism overall OB yield at ablycientffi e use of hostthe ChchNPV-TF1 cellular virus. resources for OB production by to measure productivity in production systems,cate as the PR relationship values between indi- theto amount infect of a inoculum larva required anddeath of the each number infected larva. of OBs produced following the 5.7 than the JHA-modified nucleopolyhedroviruses. hedroviruses in commercial production. instar at the moment of infectionyield, has OB/mg a marked larval influence weight onvirus total is productivity. Accordingly, a ChchNPV-TF1 productivity more per mg representativeof larval measure weight of is 53-foldsystem higher (3.42 than the matalis of ChchNPV-TF1 represents an any of the previously mentioned nucleopolyhedrovirustems. pathosys- Indeed, thethat productivity of of granuloviruses, many ChchNPV-TF1 of was which closer produce more to OBs than gemmatalis– late the PR for ChchNPV-TF1 as we did not quantify the number of exempta system (7.5 densities tested here had noweight, significant but influence on did larvalchalcites aectff body cannibalistic behavior.species, Cannibalism such in as 4 42 lar- 34–36 6 or L when www.soci.org A Bernal or the 5 26 were less 6 stage were or generate High inocu- Additionally, As such, the 6 However, this © 2018 Society of Chemical Industry As observed in 32,37 26,39 33,34 24–26 was 20- and 5-fold 40,41 By contrast, if the 22,43 OBs (Fig. 3C), similar to Spodoptera exigua 14,24,30–32 10 10 C. chalcites × 6 ,asinsectsintheL 8.15 ± resulted in similar speed of kill val- ,respectively.Larvalweightgainhas 11 5 50 10 × C. chalcites to LC and L larvae. 6 4 90 insects inoculated at 24 h post molting reported 6 38 Another important consideration for the choice of host instar ChchNPV-TF1 OB production in L The inoculum OB concentration influences overall OB produc- In the present study, the moment of inoculation was also highly stage-dependent in some species, with later instarsan often showing increasing tendency to devour conspecifics. for OB productionin is many cannibalism. species This of is Lepidoptera a during the frequent larval behavior stage and is several other host–baculovirus systems. been observed to result in exponential increases in OB yields in in the previous experiment. wileyonlinelibrary.com/journal/ps stage. A similar tendency was observed in JHA-treated larvae were compared with untreatedIn conspecifics. both species, casual observations indicated that L Other authors have made usebody of weight the and relationship OB betweenanalogues production larval (JHA) by to applying juvenile increase hormone larval weight gain on semi-synthetic diet alone orvariables gregariously. of Specifically, instar, the timing key ofand inoculation, inoculum post-inoculation concentration rearing densitycally for were theirectsff e examined on systemati- OB yields. 4DISCUSSION We describe an efficientquantities of procedure ChchNPV-TF1 OBs for using the homologous the host reared production of large alone produced 4.13 penultimate instar if the final instar is resistant to infection. increased weight atciency inoculation in 24-h-old and infected increased larvae,of in replication which OB/mg the e ffi- larval mean production weightrecently molted was L over 4-fold higher compared with vae, OB production increased 6-fold comparedresulted with the from yield the that inoculationthe of same recently instar. molted This conspecificsect eff of appears to involve a combination of less prone to cannibalistic behavior than those in the L the increase in larval weight during infection. the present study, this is dueinsect to the weight positive at relationship between deathon and the OB weight production, of which the is larvae dependent at the moment of inoculation and insect. supernumerary host instars in order to increase OB yields per tion byecting aff that speed succumb of to kill lethal and polyhedrosis the disease. proportion of insects higher than in L agile and apparently less pronetions that to earlier engage instars. in aggressive interac- influential in OB production. When inoculated as 24-h-old L ues (MTD), which allowed infected insects to reach a similar the value for L lum concentrations usuallytion, reduce as larvae the diefewer duration in OBs, of earlier because developmental theand stages infected infec- and the larvae produce virus have has less less time time to to develop replicate. was not the case in final larval instar is often selected for OB production, inoculum concentration isinfection. low, In fewer the insectsing present from acquire study, the inoculum a LC concentrations lethal rang- and sluggish responses, makingof them conspecific more predation likely than to healthy be insects. victims nucleopolyhedrovirus-infected larvae often show lower mobility

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(2013). ticidal characteristicsChrysodeixis of chalcites a nucleopolyhedrovirus isolate from for control of tomato and banana crops. in Latin America:perspectives. historical overview, current status andfor the future management of insectapplication in pests: South a Africa. focus on development and control of Lepidoptera. Simón O, A native variantdrovirus: of the Chrysodeixis basis chalcites forchalcites nucleopolyhe- a promising bioinsecticide for control of Sci D insecticides in China. the future. baculoviruses for pest control, in Pests from Theory toDiego, Practice pp. 109–124 (2017). Goettel MS, Insect pathogens as biological control agents: back to 8 Bernal A, Williams T, Caballero P and Simón O, Stage-specific insec- 7 Bernal A, Carnero A, Hernández-Suárez E, Williams T, Caballero P and 9SimónO,BernalA,WilliamsT,CarneroA,Hernández-SuárezE,Muñoz 25 Grzywacz D, Moore D and Rabindra RJ, Mass production of26 ento- Arrizubieta M, Simón O, Williams T and Caballero P, Determinant fac- 20 Sun X, Sun X, Bai B, Werf WVD,21 Vlak Claus JM JD, Gioria VV, and Miceloud GA and Hu Visnovsky G, Production Z, of insec- Production 22 Kumar CM, Sathiah N and Rabindra RJ, Optimizing the time of harvest 23 Rabindra RJ, Sathiah N, Jayanth KP and Gupta RK, Commercial scale 24 Reid S, Chan L and van Oers M, Production of entomopathogenic 27 Greene GL, Leppla NC and Dickerson WA, Velvetbean caterpillar: a rear- 19 Pijlman GP, Vrij J, van den End FJ, Vlak JM and Martens DE, Evaluation of 17 Dai X, Hajós JP, Joosten NN, van Oers MM, Ijkel WFJ, Zuidema D 18 Pijlman GP, van den Born E, Martens DE and Vlak JM, 10 Haase S, Sciocco-Cap A and Romanowski V, Baculovirus insecticides 11 Knox C, Moore SD, Luke GA and Hill MP, Baculovirus-based strategies 12 Moscardi F, Assessment of the application of baculoviruses for the 13 Sun X, History and current status of development and use of viral 14 Grzywacz D and Moore S, Production, formulation and bioassay of 15 Lacey LA, Grzywacz D, Shapiro-Ilan DI, Frutos R, Brownbridge M and 16 van Oers MM, Opportunities and challenges for the baculovirus ) ∼ 13 C. 90 10 × :21–24 225 Given this, I (Projects OB/ml (LC + 4,9 larvae would, . 8 D :460–470 (2004). 40 ha of banana 10 + Phytoma × ∼ Sanidad Vegetal en la 330 kurstaki We conclude that the Calidad y sostenibilidad en el var. C. chalcites (Esper, 1789), una plaga emer- Virology larvae with the correspond- . although how this compares 6 12,53,54 ,ed.byNoguerolesC.ASPROCAN, OB/ha, as application volumes 14 ), 12 6 10 10 × × ,ed.byMorenoR.ConsejeríadeAgricultura :e0181384 (2017). Integrated Pest Management: Dissemination and 12 :1586–1592 ©2018SocietyofChemicalIndustry Chrysodeixis chalcites 74 Chrysodeixis chalcites Bacillus thuringiensis 2018; PLoS ONE ,ed.byPeshinRandDhawanAK.Springer,Dordrecht,pp. insects with an inoculum of 9.02 -ChchNPV-TF1 pathosystem represents a remarkable 6 infestations on banana crops following applications of inoculum concentration, followed by rearing at a density Application of ChchNPV-TF1 OBs proved to be as or more OB/l, equivalent to 2 4 tification and characterization of a DNA photolyase-containingulovirus bac- from bicho camello, gente en los cultivos de platanera de Canarias. menting integrated pest managementoped countries, in in developing andImpact devel- 275–305 (2009). control de plagas ycultivo enfermedades, de la in platanera en Canarias Tenerife, pp. 145–170 (2012).en cultivos hortícolasHorticultura en Protegida invernaderos, in y Pesca, Seville, pp. 179–211 (1994). (2011). P, Chrysodeixis chalcites nucelopolyhedrovirus (ChchNPV):occurrence and natural efficacy as a biological insecticideplants on young banana in greenhouseIslands. and open-field conditions on the Canary 90 9 2000 l/ha are often required forectiveff e coverage of banana 10 ∼ Recent studies have demonstrated highly efficient control of × 1 van Oers MM, Herniou EA, Usmany M, Messelink GJ and Vlak JM, Iden- 3 Del Pino M, Carnero A, Cabello T and Hernández-Suárez E, La lagarta o 2 Shepard BM, Hammig MD, Carner GL, Ooi PAG and Smith JP, Imple- 6 Cabello T and Belda J, Noctuidos plaga (Lepidoptera: Noctuidae) 5DomínguezE,López-CeperoJandNoguerolesC,Identificacióny 4FuentesG,Hernández-SuárezE,SimónO,WilliamsTandCaballero ectiveeff for crop protection,on indoxacarb as or commercial insecticides based of 150 larvae/container resulted in the production of 8.07 with the ChchNPV-TF1 system remainstheless, to the inoculation be of determined. 24-h-old Never- L Pest Manag Sci REFERENCES and theAGL2008-05456-CO3-01 Programa and AGL2011-30352-CO2-01). Nacional Español de I assistance and insect rearing,providing and logistical Gabriel support Mercado to (INECOL)support TW. for from This the study Instituto received(Project Nacional financial de RTA2010-00016-C02-02 Investigaciones Agrarias and RTA2013-00114-C02-02) ACKNOWLEDGEMENTS We thank Noelia Gorría and Itxaso Ibáñez (UPNA) for technical chalcites 1 OBs of the ChchNPV-TF1 isolate. est PR value reported for a nucleopolyhedrovirus is thatin of its SeMNPV homologous host (1.2 ProductionofahighlyinsecticidalChchNPVisolateinoculum OBs ingested by theerent diff instars. To date, the high- www.soci.org ing LC plants. of C. chalcites opportunity for thecontrol of production this pest of in greenhouse and a field crops. biological insecticide for followed by rearing at a density of 150 larvae/container. 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