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Bionomics of Opius bellus(Hymenoptera Braconidae) anendoparasitoid of Anastrephafraterculus (Diptera Tephritidae) infruit-growing areas of NorthwesternArgentinaPablo Schlisermana Guido Alejandro Van NieuwenhoveaLaura Patricia Bezdjiana Patricia Albornoz-Medinaa LorenaIneacutes Escobara Mariacutea Josefina Buonocore Biancheria JavierAltamiranoa Martiacuten Alujab amp Sergio Marcelo Ovruskiaa Laboratorio de Investigaciones Ecoetoloacutegicas de Moscas dela Fruta y sus Enemigos Naturales (LIEMEN) Divisioacuten ControlBioloacutegico de Plagas Planta Piloto de Procesos IndustrialesMicrobioloacutegicos y Biotecnologiacutea (PROIMI) CCT Tucumaacuten CONICETAv Belgrano y Pje Caseros (T4001MVB) San Miguel de TucumaacutenArgentinab Instituto de Ecologiacutea AC Apartado Postal 63 CP 91000Xalapa Veracruz MeacutexicoAccepted author version posted online 08 Nov 2013Publishedonline 10 Mar 2014

To cite this article Pablo Schliserman Guido Alejandro Van Nieuwenhove Laura PatriciaBezdjian Patricia Albornoz-Medina Lorena Ineacutes Escobar Mariacutea Josefina Buonocore BiancheriJavier Altamirano Martiacuten Aluja amp Sergio Marcelo Ovruski (2014) Bionomics of Opius bellus(Hymenoptera Braconidae) an endoparasitoid of Anastrepha fraterculus (Diptera Tephritidae) infruit-growing areas of Northwestern Argentina Biocontrol Science and Technology 244 375-388DOI 101080095831572013863826

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RESEARCH ARTICLE

Bionomics of Opius bellus (Hymenoptera Braconidae) anendoparasitoid of Anastrepha fraterculus (Diptera Tephritidae) in fruit-

growing areas of Northwestern Argentina

Pablo Schlisermana Guido Alejandro Van Nieuwenhovea Laura Patricia BezdjianaPatricia Albornoz-Medinaa Lorena Ineacutes Escobara

Mariacutea Josefina Buonocore Biancheria Javier Altamiranoa Martiacuten Alujab andSergio Marcelo Ovruskia

aLaboratorio de Investigaciones Ecoetoloacutegicas de Moscas de la Fruta y sus Enemigos Naturales(LIEMEN) Divisioacuten Control Bioloacutegico de Plagas Planta Piloto de Procesos Industriales

Microbioloacutegicos y Biotecnologiacutea (PROIMI) CCT Tucumaacuten CONICET Av Belgrano y PjeCaseros (T4001MVB) San Miguel de Tucumaacuten Argentina bInstituto de Ecologiacutea AC

Apartado Postal 63 CP 91000 Xalapa Veracruz Meacutexico

(Received 28 February 2013 returned 24 April 2013 accepted 4 November 2013)

Opius bellus is a neotropical larval-prepupal parasitoid known to attack thepestiferous fruit fly Anastrepha fraterculus Due to interest in the use of nativeparasitoids in forthcoming fruit fly biocontrol programmes in Argentina O belluswas colonised for the first time using laboratory-reared A fraterculus larvae Aseries of experiments were conducted to (1) best achieve an efficient parasitoidrearing by determining optimal larval host age hostparasitoid ratio and hostexposure time and (2) assess their potential as biological control agents bydetermining reproductive parameters The most productive exposure regimen was7ndash9 d-old (early and middle third-instars) A fraterculus larvae for 4 h at a 41 hostparasitoid ratio this array of factors was sufficient to achieve the highest averageadult emergence (48) and an offspring sex ratio at equitable proportionIncreasing both hostparasitoid ratio further than 41 and the host exposuretime beyond 4 h did not significantly enhance parasitoid female offspring yieldFemales produced eggs for 295 plusmn 14 days At 32 days of age 50 of the femaleswere still alive The majority of the progeny were produced by females between 20and 24 d-old At 26degC gross fecundity rate net reproductive rate intrinsic rate ofincrease and mean generation time were 207 plusmn 42 offspringfemale 96 plusmn 25femalesnewborn females 006 plusmn 001 femalesfemaleday and 84 plusmn 02 daysrespectively The long lifespan and reproductive parameters suggest that thisparasitoid species has suitable attributes for mass-rearing

Keywords fruit fly parasitoid Braconidae rearing demographic parametersArgentina

Introduction

The native South American fruit fly Anastrepha fraterculus (Wiedemann) and theexotic Mediterranean fruit fly Ceratitis capitata (Wiedemann) are among the mostimportant pests affecting commercial fruit production in Argentina (Guilleacuten amp

Corresponding author Emails sovruskiproimiorgar ovruskisergioyahoocomar

Biocontrol Science and Technology 2014Vol 24 No 4 375ndash388 httpdxdoiorg101080095831572013863826

copy 2014 Taylor amp Francis

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Saacutenchez 2007) For example in the citrus-growing areas of northern Argentinaapproximately 143000 tonnes of citrus is lost every year due to A fraterculus andC capitata In addition both tephritid species severely limit the export of fruitbecause of quarantine restrictions imposed by countries free of these pests (Guilleacuten ampSaacutenchez 2007)

Interest in an integrated approach to control both pests in Argentina has resultedin attempts to apply biological control strategies These programmes were based onthe use of exotic egg larval-pupal andor pupal parasitoids that had first beenestablished in Hawaii Examples include Fopius arisanus Sonan Diachasmimorphalongicaudata (Ashmead) (Braconidae) Aceratoneuromyia indica (Silvestri) (Eulophi-dae) and Pachycrepoideus vindemiae (Rondani) (Pteromalidae) which were intro-duced and released in the early 1960s (Ovruski Aluja Sivinski amp Wharton 2000)Mass releases of the Indo-Pacific species D longicaudata a larval-prepupalendoparasitoid of several pestiferous fruit flies (Vargas et al 2002) were recentlyperformed on commercial fig crops in rural areas of San Juan (south-westernArgentina) as part of an integrated C capitata management programme on an area-wide basis (Ovruski amp Schliserman 2012) However the integrated management ofA fraterculus in Argentinarsquos northwestern citrus-growing areas might include theimplementation of augmentative releases of native parasitoids (Aluja amp Rull 2009)as well as their conservation (Jonsson Wratten Landis amp Gurr 2008)

Knowledge of the basic biology and ecology of native Anastrepha parasitoids iscritical during the selection of a candidate for augmentative release programmes inparticular environmental conditions and with unique host-densities (Sivinski Pintildeeroamp Aluja 2000) Among the factors important to the selection of biological controlagents are interactions within local parasitoid guilds (Sivinski Aluja amp Loacutepez1997) the type of host fruit attacked by the pest (Sivinski Vulinec amp Aluja 2001)the ability of the parasitoid to respond to varying densities of the host fly (Garciacutea-Medel Sivinski Diacuteaz-Fleischer Ramirez-Romero amp Aluja 2007) the parasitoiddemographic parameters and the feasibility of mass-rearing (Aluja et al 2009) Theuse of native parasitoids in a biological control programme provides substantialbenefits by avoiding costly trips abroad in search of exotic species importation andquarantine protocols and potential non-target effects on local fauna (Gateset al 2002)

Opius bellus Gahan is a wide-spread neotropical parasitoid It is a solitary larval-prepupal endoparasitic koinobiont (Ovruski et al 2000) that has one of the shortestovipositors of any native Anastrepha opiine parasitoids (Aguiar-Menezes ampMenezes 2001 Ovruski Schliserman amp Aluja 2004) It attacks 13 species ofAnastrepha and C capitata and has been recovered in 26 host plant species fromMexico (Hernaacutendez-Ortiz Gonzaacutelez Escalante-Tio amp Manrique-Saide 2006)Panama (Medianero Korytkowski Campo amp de Leoacuten 2006) Trinidad (Whartonamp Marsh 1978) Venezuela (Katiyar Camacho Geraud amp Matheus 1995) Brazil(Aguiar-Menezes amp Menezes 2001 Uchocirca-Fernandez et al 2003 De Jesuacutes et al2008) Bolivia (Ovruski et al 2009) and Argentina (Ovruski amp Schliserman 2012)However lsquoO bellusrsquo probably represents a group of cryptic species that are verydifficult to distinguish morphologically Future genetic analyses andor crossingexperiments will be needed to establish differences (Wharton 1997 Canal ampZucchi 2000)

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Since many vegetative environments are rapidly disappearing in the highlyendangered subtropical forest of Northwestern Argentina there is an urgent need tofind and preserve native parasitoid species associated with A fraterculus Conse-quently Argentinean populations of O bellus were colonised in the laboratory forthe first time The aim of the current study was to describe the demographicparameters of O bellus and identify optimal host-exposure regimens for rearingLaboratory assays were conducted using a wild O bellus strain colonised on Afraterculus to determine (1) the best host larval age (2) the best ratio of host larvaeto female parasitoids (3) the best exposure time to achieve the greatest parasitoidyield with the highest female-biased progeny ratio (4) daily survival and fecundity offemales (5) daily parasitism and adult emergence rates and (6) reproductiveparameters These findings ultimately reflect on the suitability of O bellus foraugmentative release

Materials and methodsSource of insects and laboratory colonisation procedures

The laboratory colony of O bellus was started in the insectary of the PROIMIBiological Control Division Tucumaacuten Argentina between March and April 2006with insects obtained from ripe guavas collected within wild vegetation patches inHorco Molle Tucumaacuten This parental colony was initiated by holding 38 femaleand 45 male O bellus that emerged from approximately 5000 puparia of wildA fraterculus in cubical plexiglass cages (30 cm) covered with organdy cloth on twoopposite sides provided with water and honey every other day The colony was keptin a room at 25 plusmn 1degC 75 plusmn 5 relative humidity (RH) and a 1212-h (LD)photoperiod A separate room maintained at 26 plusmn 1degC 65 plusmn 10 RH and a 1212-h(LD) photoperiod was used to rear A fraterculus

The initial step in the colonisation process was to simulate a naturally infestedfruit Roughly 500 third-instar A fraterculus larvae (8ndash11 d-old) were removed froman artificial diet and placed naked (without a diet) inside either a hollow uninfestedlsquoferalrsquo guava or a commercially grown peach fruit The procedure for filling fruitwith larvae was as described by Aluja et al 2009 Inoculated fruits were thenindividually placed within the plexiglass rearing cage and exposed to the parentalgeneration of O bellus for 24 h Infested fruits were exposed daily to parasitoids overtheir entire adult lifespan After the exposure period each infested fruit was placed ina plastic container (500 ml) with sterilised vermiculite on the bottom as a pupationsubstrate and covered with a piece of organdy cloth on the top Pupae obtained fromindividual fruit were sifted from the pupation medium and kept in plastic cups withnew sterilised moist vermiculite until all of the flies and parasitoids emerged

Once the parasitoids had reproduced for two generations using the artificiallyinfested fruit method the second step in the O bellus colonisation process was toadapt adult females to a non-natural parasitisation substrate For that an artificialoviposition unit consisting of a plastic ring (98 cm 1 cm) covered with a 13-cm pieceof organdy was built On the cloth surface ca 500 A fraterculus third-instar larvaewere placed mixed with either guava or peach pulp Fruit pulp was used because thefruit aroma attracts parasitoid females to the artificial oviposition devices (EitamHoller Sivinski amp Aluja 2003) The cloth containing the larvae and fruit pulp wasthen covered tightly with another 13-cm piece of organdy attached to the first ring by

Biocontrol Science and Technology 377

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another plastic ring (10 cm 1 cm) by pressing the cloth against the smaller ringAfter this several slices of either guava or peach epicarp (ca 1 mm in thickness) wereplaced on top of the organdy cover (Aluja et al 2009) The artificial oviposition unitwas then elevated by placing it onto an inverted plastic device (3 cm diameter 4 cmheight) to place it closer to the centre of the parasitoid rearing cage After 24 h ofexposure to parasitoids host larvae were removed from the fruit pulp andtransferred to plastic cups with sterilised and moistened vermiculite as a pupationsubstrate Adult flies and parasitoids were allowed to emerge inside these cupsEmerged parasitoids were then transferred to a new Plexiglas rearing cage

The rearing technique described above was used for three parasitoid generationsand a variant of this method was employed during the third step of the O belluscolonisation procedure The artificial parasitisation unit was the same as describedabove except that for this set-up the host larvae were exposed without fruit pulpalthough either guava or peach skin layers were still held on top of the organdy clothcover After the O bellus colony had gone through six generations of lab rearing thefruit skin pieces were also eliminated from the top of oviposition unit During thisfourth step of the O bellus colonisation process the oviposition unit was filled withca 500 A fraterculus third-instar larvae and artificial diet (brewer yeast wheat germsugar agar and water) The oviposition unit was exposed to 100 parasitoid femalesfor 8 h inside a rearing cage When the O bellus colony reached 13 generations thehost larvae exposure time was changed to 6 h All of the O bellus colonisationphases and experiments described below were performed under similar previouslydescribed environmental conditions

Optimal host larval age for parasitisation

Five age ranges of A fraterculus larvae were analysed to determine the optimallarval age to expose the host to parasitoids for oviposition 1ndash3 d-old (first instars =L1) 4ndash6 d-old ( second instars = L2) 7ndash8 d-old (early third-instars = L3E) 9ndash10 d-old(middle third-instars = L3M) and 11ndash12 d-old (late third-instars = L3L) Anastrephafraterculus larval instars were determined based on the size and shape of thecephalopharyngeal skeleton under a stereomicroscope as described by FriasSelivon amp Hernaacutendez-Ortiz (2008) For each age range 75 laboratory-reared larvaemixed with the diet on which they had been reared were exposed to 15 matedO bellus females for 24 h inside an oviposition unit placed on the floor of a cubicalplexiglass cage (15 cm) Parasitoid females were 10 d-old with no prior ovipositionexperience and from a colony that was 14 generations old Additional preliminarytests indicated that O bellus females aged 10ndash12 d yielded the highest parasitoidemergence rate (Laura Patricia Bezdjian unpub data) After exposure to para-sitoids most of the larvae were transferred into plastic trays (100 ml) filled with afresh diet medium These trays were then placed inside a cubical plastic container(25 cm) with a 2-cm layer of vermiculite on the bottom and covered with organdyEvery seven days puparia were sifted from the pupation medium and held in plasticcups (8 cm diameter 5 cm height) with fresh moist vermiculite until eclosion The11ndash12 d-old larvae were directly transferred to vermiculite inside plastic cups Thenumber of dead host larvae and the number of recovered puparia were recorded forall of the age ranges At the time of eclosion the number and sex of parasitoidprogeny and the number of non-eclosed puparia were also recorded Control tests

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(host larvae not exposed to parasitoids) were conducted for each larval age range todetermine the natural A fraterculus mortality rate Each test (including eachcontrol) was replicated 10 times Prior to the beginning of each replicate thequality of the A fraterculus larvae was assessed All the batches of host larvae withfly emergence percentages lt 90 were discarded and were not used in theexperiments

Optimal exposure time and ratio of hosts to parasitoids

To determine the optimal proportion of A fraterculus larvae per O bellus femaleand optimal amount of time to expose host larvae to parasitoids for rearing fivehostparasitoid ratios were individually analysed 21 41 61 81 and 101 Fivemated 10 d-old O bellus females that had prior oviposition experience and from acolony that was 14 generations old were used for each hostparasitoid ratiotreatment Each oviposition unit was filled with 7ndash9 d-old host larvae plus freshlarval diet Hosts at each density were exposed to parasitoid females in a separatecubical Plexiglas cages (20 cm) for 1 2 3 4 and 5 h After exposure to parasitoidslarvae were transferred into plastic trays and processed as described above After 2 ddead larvae were removed from trays and counted Also pupae were removed fromthe pupation medium on a daily basis

Basic biological and demographic parameters

To determine the demographic parameters of O bellus four groups with five pairsof newly emerged (lt24 h old) adult parasitoids in each group were placedindividually in cubical plexiglass cages (20 cm) and provided daily with ca A totalof 100 laboratory-reared 7ndash9 d-old A fraterculus larvae inside an oviposition unitplaced on the floor of the cage Water and honey were provided to the parasitoidsevery other day After a 4-h exposure to parasitoid females host larvae wereremoved and placed into plastic cups (8 cm diameter 5 cm height) with sterilisedvermiculite Pupae were held in these cups for five weeks to allow for adultemergence Emerged adult flies and parasitoids were taken out of each cup everyday counted and sexed The remaining non-eclosed puparia were dissected to checkfor the presence of parasitoid cadavers Wasprsquos mortality was recorded and the deadwasps were removed from each test cage on daily basis The assay was continueduntil all of the parasitoids died The basic biological parameters such as the pre-oviposition and oviposition periods adult lifespan development time (from egg toadult) offspring sex ratios (measured as the female proportion) and daily parasitismand emergence rates were determined for the O bellus cohort Furthermorestandard life table parameters (Carey 1993) were determined from the daily recordsof mortality and birth (Vargas et al 2002) The daily proportion of survivingparental females (lx) and the daily female offspring produced per female (mx) wereestimated and illustrated From the life table the following demographic parameterswere calculated according to formulae used by Carey (1993) gross fecundity rate netfecundity rate gross reproductive rate net reproductive rate intrinsic rate ofincrease finite rate of increase and mean generation time

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Data analysis

Parasitism was estimated as the total number of emerged and non-emergedparasitoids divided by the total number of obtained pupae and multiplied by 100whereas parasitoid emergence was calculated as the total number of emergedoffspring divided by the total number of recovered pupae and multiplied by 100Host mortality was estimated as the total number of dead larvae plus the totalnumber of puparia that did not yield insects divided by the total number of exposedhost larvae and multiplied by 100 The data regarding parasitoid emergenceparasitoid female offspring proportion (sex ratio) and host mortality were comparedthrough univariate General Linear Models (GLM) with a significance threshold ofP = 005 (Zar 1999) Mean comparisons were analysed by Tukeyrsquos honestysignificant difference (HSD) test at P = 005 Host mortality recorded from eachhost larval age trial was compared with its respective control through a t-test atP = 005 All of the proportional data were arcsine square root-transformed prior toanalysis Only the untransformed means (plusmn SE) are presented in the text

Results

A significant effect of host larval development stage on each response variable wasfound (F(415) = 42843 P lt 00001 for parasitoid emergence F(415) = 26782P lt 00001 for sex ratio F(415) = 10684 P lt 00001 for host mortality) Third-instarlarvae were most parasitised and produced a higher percentage of emerged O bellusand female offspring than those of the first and second instars (Figure 1) L3E and

Figure 1 Influence of A fraterculus instars on the percentages (mean plusmn SE) of emergedO bellus adults parasitoid female offspring (sex ratio) and host larval and pupal mortalityrecorded in optimal host larval-age tests Bars followed by the same letter indicate nosignificant differences (Tukey HSD test P = 005) Notations L1 first instars L2 secondinstars L3E early third-instars L3M middle third-instars L3L late third-instars

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L3M had the highest adult emergence values while both larval instars andL3L yielded statistically similar sex ratios (Figure 1) Among the third-instar larvaethe lowest host mortality percentage was recorded on L3E but was comparable tothat recorded from L1 (Figure 1) Host mortality recorded from L1 L2 L3E L3M andL3L was 25- 39- 49- 42- and 71-fold significantly higher than mortality recordedfrom their respective controls (t = 601 t = 942 t = 107 t = 583 t =114 for L1 L2L3E L3M and L3L respectively df = 18 and P lt 00001 for all five host age classes)

Both exposure time and ratio of hosts to parasitoids and their interaction had asignificant effect on parasitoid emergence and host mortality but not on the sex ratioof parasitoid (Table 1) Parasitoid emergence significantly increased at 41 61 81and 101 hostparasitoid ratios (Figure 2) and over the longer host larvae exposuretimes (4 and 5 h) (Figure 3) Percentage of parasitoid female offspring wassignificantly similar among all treatments and was generally male-biased Only at41 hostparasitoid ratio (Figure 2) and at 4 h exposure time (Figure 3) did O bellusexhibit a relatively female-biased sex ratio (1041 femalesmale) Host mortality waslower at a 21 hostparasitoid ratio (Figure 2) and over the shorter host exposuretimes (1ndash3 h) (Figure 3)

The pre-imaginal development times for male and female O bellus were 201 plusmn14 (mean plusmn standard error) and 216 plusmn 02 days at 25degC respectively A short pre-oviposition period (20 plusmn 06 days) was observed for mated females which producedeggs for 295 plusmn 14 days on average The mean female longevity was 303 plusmn 24 dayswhereas the mean male longevity was 274 plusmn 14 days At 32 days of age 50 of thefemales were still alive but at 60 d-old less than 10 of the females remained alive(Figure 4) The daily production of female offspring per parental female increaseduntil day 23 of the female reproductive life (23 plusmn 04 daughters per female) anddecreased thereafter (Figure 4)

The lifetime production of offspring (males plus females) of an average parentfemale was 207 plusmn 42 and the mean sex ratio (proportion of females) of the progenywas 580 plusmn 03 The net fecundity rate was 161 plusmn 67 offspring per newborn femaleThe mean number of female offspring produced by a single female during her

Table 1 Summary of univariate GLMs on the effect of host exposure time ratio of hosts toparasitoids and interactions of these categorical factors on parasitoid emergence parasitoidprogeny sex ratio and host mortality

Response variables

Parasitoidemergence

Parasitoidoffspring sex

ratio Host mortality

Source of variation df Error df F P F P F P

Hostparasitoidratio (HPR)

4 100 992 lt 00001a 038 = 08186 1525 lt 00001a

Host exposuretime (HET)

4 100 9154 lt 00001a 096 = 04319 276 = 00319a

HPR times HET 16 100 210 = 00137a 063 = 08454 440 lt 00001a

aSignificant variables

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

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Since many vegetative environments are rapidly disappearing in the highlyendangered subtropical forest of Northwestern Argentina there is an urgent need tofind and preserve native parasitoid species associated with A fraterculus Conse-quently Argentinean populations of O bellus were colonised in the laboratory forthe first time The aim of the current study was to describe the demographicparameters of O bellus and identify optimal host-exposure regimens for rearingLaboratory assays were conducted using a wild O bellus strain colonised on Afraterculus to determine (1) the best host larval age (2) the best ratio of host larvaeto female parasitoids (3) the best exposure time to achieve the greatest parasitoidyield with the highest female-biased progeny ratio (4) daily survival and fecundity offemales (5) daily parasitism and adult emergence rates and (6) reproductiveparameters These findings ultimately reflect on the suitability of O bellus foraugmentative release

Materials and methodsSource of insects and laboratory colonisation procedures

The laboratory colony of O bellus was started in the insectary of the PROIMIBiological Control Division Tucumaacuten Argentina between March and April 2006with insects obtained from ripe guavas collected within wild vegetation patches inHorco Molle Tucumaacuten This parental colony was initiated by holding 38 femaleand 45 male O bellus that emerged from approximately 5000 puparia of wildA fraterculus in cubical plexiglass cages (30 cm) covered with organdy cloth on twoopposite sides provided with water and honey every other day The colony was keptin a room at 25 plusmn 1degC 75 plusmn 5 relative humidity (RH) and a 1212-h (LD)photoperiod A separate room maintained at 26 plusmn 1degC 65 plusmn 10 RH and a 1212-h(LD) photoperiod was used to rear A fraterculus

The initial step in the colonisation process was to simulate a naturally infestedfruit Roughly 500 third-instar A fraterculus larvae (8ndash11 d-old) were removed froman artificial diet and placed naked (without a diet) inside either a hollow uninfestedlsquoferalrsquo guava or a commercially grown peach fruit The procedure for filling fruitwith larvae was as described by Aluja et al 2009 Inoculated fruits were thenindividually placed within the plexiglass rearing cage and exposed to the parentalgeneration of O bellus for 24 h Infested fruits were exposed daily to parasitoids overtheir entire adult lifespan After the exposure period each infested fruit was placed ina plastic container (500 ml) with sterilised vermiculite on the bottom as a pupationsubstrate and covered with a piece of organdy cloth on the top Pupae obtained fromindividual fruit were sifted from the pupation medium and kept in plastic cups withnew sterilised moist vermiculite until all of the flies and parasitoids emerged

Once the parasitoids had reproduced for two generations using the artificiallyinfested fruit method the second step in the O bellus colonisation process was toadapt adult females to a non-natural parasitisation substrate For that an artificialoviposition unit consisting of a plastic ring (98 cm 1 cm) covered with a 13-cm pieceof organdy was built On the cloth surface ca 500 A fraterculus third-instar larvaewere placed mixed with either guava or peach pulp Fruit pulp was used because thefruit aroma attracts parasitoid females to the artificial oviposition devices (EitamHoller Sivinski amp Aluja 2003) The cloth containing the larvae and fruit pulp wasthen covered tightly with another 13-cm piece of organdy attached to the first ring by

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another plastic ring (10 cm 1 cm) by pressing the cloth against the smaller ringAfter this several slices of either guava or peach epicarp (ca 1 mm in thickness) wereplaced on top of the organdy cover (Aluja et al 2009) The artificial oviposition unitwas then elevated by placing it onto an inverted plastic device (3 cm diameter 4 cmheight) to place it closer to the centre of the parasitoid rearing cage After 24 h ofexposure to parasitoids host larvae were removed from the fruit pulp andtransferred to plastic cups with sterilised and moistened vermiculite as a pupationsubstrate Adult flies and parasitoids were allowed to emerge inside these cupsEmerged parasitoids were then transferred to a new Plexiglas rearing cage

The rearing technique described above was used for three parasitoid generationsand a variant of this method was employed during the third step of the O belluscolonisation procedure The artificial parasitisation unit was the same as describedabove except that for this set-up the host larvae were exposed without fruit pulpalthough either guava or peach skin layers were still held on top of the organdy clothcover After the O bellus colony had gone through six generations of lab rearing thefruit skin pieces were also eliminated from the top of oviposition unit During thisfourth step of the O bellus colonisation process the oviposition unit was filled withca 500 A fraterculus third-instar larvae and artificial diet (brewer yeast wheat germsugar agar and water) The oviposition unit was exposed to 100 parasitoid femalesfor 8 h inside a rearing cage When the O bellus colony reached 13 generations thehost larvae exposure time was changed to 6 h All of the O bellus colonisationphases and experiments described below were performed under similar previouslydescribed environmental conditions

Optimal host larval age for parasitisation

Five age ranges of A fraterculus larvae were analysed to determine the optimallarval age to expose the host to parasitoids for oviposition 1ndash3 d-old (first instars =L1) 4ndash6 d-old ( second instars = L2) 7ndash8 d-old (early third-instars = L3E) 9ndash10 d-old(middle third-instars = L3M) and 11ndash12 d-old (late third-instars = L3L) Anastrephafraterculus larval instars were determined based on the size and shape of thecephalopharyngeal skeleton under a stereomicroscope as described by FriasSelivon amp Hernaacutendez-Ortiz (2008) For each age range 75 laboratory-reared larvaemixed with the diet on which they had been reared were exposed to 15 matedO bellus females for 24 h inside an oviposition unit placed on the floor of a cubicalplexiglass cage (15 cm) Parasitoid females were 10 d-old with no prior ovipositionexperience and from a colony that was 14 generations old Additional preliminarytests indicated that O bellus females aged 10ndash12 d yielded the highest parasitoidemergence rate (Laura Patricia Bezdjian unpub data) After exposure to para-sitoids most of the larvae were transferred into plastic trays (100 ml) filled with afresh diet medium These trays were then placed inside a cubical plastic container(25 cm) with a 2-cm layer of vermiculite on the bottom and covered with organdyEvery seven days puparia were sifted from the pupation medium and held in plasticcups (8 cm diameter 5 cm height) with fresh moist vermiculite until eclosion The11ndash12 d-old larvae were directly transferred to vermiculite inside plastic cups Thenumber of dead host larvae and the number of recovered puparia were recorded forall of the age ranges At the time of eclosion the number and sex of parasitoidprogeny and the number of non-eclosed puparia were also recorded Control tests

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(host larvae not exposed to parasitoids) were conducted for each larval age range todetermine the natural A fraterculus mortality rate Each test (including eachcontrol) was replicated 10 times Prior to the beginning of each replicate thequality of the A fraterculus larvae was assessed All the batches of host larvae withfly emergence percentages lt 90 were discarded and were not used in theexperiments

Optimal exposure time and ratio of hosts to parasitoids

To determine the optimal proportion of A fraterculus larvae per O bellus femaleand optimal amount of time to expose host larvae to parasitoids for rearing fivehostparasitoid ratios were individually analysed 21 41 61 81 and 101 Fivemated 10 d-old O bellus females that had prior oviposition experience and from acolony that was 14 generations old were used for each hostparasitoid ratiotreatment Each oviposition unit was filled with 7ndash9 d-old host larvae plus freshlarval diet Hosts at each density were exposed to parasitoid females in a separatecubical Plexiglas cages (20 cm) for 1 2 3 4 and 5 h After exposure to parasitoidslarvae were transferred into plastic trays and processed as described above After 2 ddead larvae were removed from trays and counted Also pupae were removed fromthe pupation medium on a daily basis

Basic biological and demographic parameters

To determine the demographic parameters of O bellus four groups with five pairsof newly emerged (lt24 h old) adult parasitoids in each group were placedindividually in cubical plexiglass cages (20 cm) and provided daily with ca A totalof 100 laboratory-reared 7ndash9 d-old A fraterculus larvae inside an oviposition unitplaced on the floor of the cage Water and honey were provided to the parasitoidsevery other day After a 4-h exposure to parasitoid females host larvae wereremoved and placed into plastic cups (8 cm diameter 5 cm height) with sterilisedvermiculite Pupae were held in these cups for five weeks to allow for adultemergence Emerged adult flies and parasitoids were taken out of each cup everyday counted and sexed The remaining non-eclosed puparia were dissected to checkfor the presence of parasitoid cadavers Wasprsquos mortality was recorded and the deadwasps were removed from each test cage on daily basis The assay was continueduntil all of the parasitoids died The basic biological parameters such as the pre-oviposition and oviposition periods adult lifespan development time (from egg toadult) offspring sex ratios (measured as the female proportion) and daily parasitismand emergence rates were determined for the O bellus cohort Furthermorestandard life table parameters (Carey 1993) were determined from the daily recordsof mortality and birth (Vargas et al 2002) The daily proportion of survivingparental females (lx) and the daily female offspring produced per female (mx) wereestimated and illustrated From the life table the following demographic parameterswere calculated according to formulae used by Carey (1993) gross fecundity rate netfecundity rate gross reproductive rate net reproductive rate intrinsic rate ofincrease finite rate of increase and mean generation time

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Data analysis

Parasitism was estimated as the total number of emerged and non-emergedparasitoids divided by the total number of obtained pupae and multiplied by 100whereas parasitoid emergence was calculated as the total number of emergedoffspring divided by the total number of recovered pupae and multiplied by 100Host mortality was estimated as the total number of dead larvae plus the totalnumber of puparia that did not yield insects divided by the total number of exposedhost larvae and multiplied by 100 The data regarding parasitoid emergenceparasitoid female offspring proportion (sex ratio) and host mortality were comparedthrough univariate General Linear Models (GLM) with a significance threshold ofP = 005 (Zar 1999) Mean comparisons were analysed by Tukeyrsquos honestysignificant difference (HSD) test at P = 005 Host mortality recorded from eachhost larval age trial was compared with its respective control through a t-test atP = 005 All of the proportional data were arcsine square root-transformed prior toanalysis Only the untransformed means (plusmn SE) are presented in the text

Results

A significant effect of host larval development stage on each response variable wasfound (F(415) = 42843 P lt 00001 for parasitoid emergence F(415) = 26782P lt 00001 for sex ratio F(415) = 10684 P lt 00001 for host mortality) Third-instarlarvae were most parasitised and produced a higher percentage of emerged O bellusand female offspring than those of the first and second instars (Figure 1) L3E and

Figure 1 Influence of A fraterculus instars on the percentages (mean plusmn SE) of emergedO bellus adults parasitoid female offspring (sex ratio) and host larval and pupal mortalityrecorded in optimal host larval-age tests Bars followed by the same letter indicate nosignificant differences (Tukey HSD test P = 005) Notations L1 first instars L2 secondinstars L3E early third-instars L3M middle third-instars L3L late third-instars

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L3M had the highest adult emergence values while both larval instars andL3L yielded statistically similar sex ratios (Figure 1) Among the third-instar larvaethe lowest host mortality percentage was recorded on L3E but was comparable tothat recorded from L1 (Figure 1) Host mortality recorded from L1 L2 L3E L3M andL3L was 25- 39- 49- 42- and 71-fold significantly higher than mortality recordedfrom their respective controls (t = 601 t = 942 t = 107 t = 583 t =114 for L1 L2L3E L3M and L3L respectively df = 18 and P lt 00001 for all five host age classes)

Both exposure time and ratio of hosts to parasitoids and their interaction had asignificant effect on parasitoid emergence and host mortality but not on the sex ratioof parasitoid (Table 1) Parasitoid emergence significantly increased at 41 61 81and 101 hostparasitoid ratios (Figure 2) and over the longer host larvae exposuretimes (4 and 5 h) (Figure 3) Percentage of parasitoid female offspring wassignificantly similar among all treatments and was generally male-biased Only at41 hostparasitoid ratio (Figure 2) and at 4 h exposure time (Figure 3) did O bellusexhibit a relatively female-biased sex ratio (1041 femalesmale) Host mortality waslower at a 21 hostparasitoid ratio (Figure 2) and over the shorter host exposuretimes (1ndash3 h) (Figure 3)

The pre-imaginal development times for male and female O bellus were 201 plusmn14 (mean plusmn standard error) and 216 plusmn 02 days at 25degC respectively A short pre-oviposition period (20 plusmn 06 days) was observed for mated females which producedeggs for 295 plusmn 14 days on average The mean female longevity was 303 plusmn 24 dayswhereas the mean male longevity was 274 plusmn 14 days At 32 days of age 50 of thefemales were still alive but at 60 d-old less than 10 of the females remained alive(Figure 4) The daily production of female offspring per parental female increaseduntil day 23 of the female reproductive life (23 plusmn 04 daughters per female) anddecreased thereafter (Figure 4)

The lifetime production of offspring (males plus females) of an average parentfemale was 207 plusmn 42 and the mean sex ratio (proportion of females) of the progenywas 580 plusmn 03 The net fecundity rate was 161 plusmn 67 offspring per newborn femaleThe mean number of female offspring produced by a single female during her

Table 1 Summary of univariate GLMs on the effect of host exposure time ratio of hosts toparasitoids and interactions of these categorical factors on parasitoid emergence parasitoidprogeny sex ratio and host mortality

Response variables

Parasitoidemergence

Parasitoidoffspring sex

ratio Host mortality

Source of variation df Error df F P F P F P

Hostparasitoidratio (HPR)

4 100 992 lt 00001a 038 = 08186 1525 lt 00001a

Host exposuretime (HET)

4 100 9154 lt 00001a 096 = 04319 276 = 00319a

HPR times HET 16 100 210 = 00137a 063 = 08454 440 lt 00001a

aSignificant variables

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

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another plastic ring (10 cm 1 cm) by pressing the cloth against the smaller ringAfter this several slices of either guava or peach epicarp (ca 1 mm in thickness) wereplaced on top of the organdy cover (Aluja et al 2009) The artificial oviposition unitwas then elevated by placing it onto an inverted plastic device (3 cm diameter 4 cmheight) to place it closer to the centre of the parasitoid rearing cage After 24 h ofexposure to parasitoids host larvae were removed from the fruit pulp andtransferred to plastic cups with sterilised and moistened vermiculite as a pupationsubstrate Adult flies and parasitoids were allowed to emerge inside these cupsEmerged parasitoids were then transferred to a new Plexiglas rearing cage

The rearing technique described above was used for three parasitoid generationsand a variant of this method was employed during the third step of the O belluscolonisation procedure The artificial parasitisation unit was the same as describedabove except that for this set-up the host larvae were exposed without fruit pulpalthough either guava or peach skin layers were still held on top of the organdy clothcover After the O bellus colony had gone through six generations of lab rearing thefruit skin pieces were also eliminated from the top of oviposition unit During thisfourth step of the O bellus colonisation process the oviposition unit was filled withca 500 A fraterculus third-instar larvae and artificial diet (brewer yeast wheat germsugar agar and water) The oviposition unit was exposed to 100 parasitoid femalesfor 8 h inside a rearing cage When the O bellus colony reached 13 generations thehost larvae exposure time was changed to 6 h All of the O bellus colonisationphases and experiments described below were performed under similar previouslydescribed environmental conditions

Optimal host larval age for parasitisation

Five age ranges of A fraterculus larvae were analysed to determine the optimallarval age to expose the host to parasitoids for oviposition 1ndash3 d-old (first instars =L1) 4ndash6 d-old ( second instars = L2) 7ndash8 d-old (early third-instars = L3E) 9ndash10 d-old(middle third-instars = L3M) and 11ndash12 d-old (late third-instars = L3L) Anastrephafraterculus larval instars were determined based on the size and shape of thecephalopharyngeal skeleton under a stereomicroscope as described by FriasSelivon amp Hernaacutendez-Ortiz (2008) For each age range 75 laboratory-reared larvaemixed with the diet on which they had been reared were exposed to 15 matedO bellus females for 24 h inside an oviposition unit placed on the floor of a cubicalplexiglass cage (15 cm) Parasitoid females were 10 d-old with no prior ovipositionexperience and from a colony that was 14 generations old Additional preliminarytests indicated that O bellus females aged 10ndash12 d yielded the highest parasitoidemergence rate (Laura Patricia Bezdjian unpub data) After exposure to para-sitoids most of the larvae were transferred into plastic trays (100 ml) filled with afresh diet medium These trays were then placed inside a cubical plastic container(25 cm) with a 2-cm layer of vermiculite on the bottom and covered with organdyEvery seven days puparia were sifted from the pupation medium and held in plasticcups (8 cm diameter 5 cm height) with fresh moist vermiculite until eclosion The11ndash12 d-old larvae were directly transferred to vermiculite inside plastic cups Thenumber of dead host larvae and the number of recovered puparia were recorded forall of the age ranges At the time of eclosion the number and sex of parasitoidprogeny and the number of non-eclosed puparia were also recorded Control tests

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(host larvae not exposed to parasitoids) were conducted for each larval age range todetermine the natural A fraterculus mortality rate Each test (including eachcontrol) was replicated 10 times Prior to the beginning of each replicate thequality of the A fraterculus larvae was assessed All the batches of host larvae withfly emergence percentages lt 90 were discarded and were not used in theexperiments

Optimal exposure time and ratio of hosts to parasitoids

To determine the optimal proportion of A fraterculus larvae per O bellus femaleand optimal amount of time to expose host larvae to parasitoids for rearing fivehostparasitoid ratios were individually analysed 21 41 61 81 and 101 Fivemated 10 d-old O bellus females that had prior oviposition experience and from acolony that was 14 generations old were used for each hostparasitoid ratiotreatment Each oviposition unit was filled with 7ndash9 d-old host larvae plus freshlarval diet Hosts at each density were exposed to parasitoid females in a separatecubical Plexiglas cages (20 cm) for 1 2 3 4 and 5 h After exposure to parasitoidslarvae were transferred into plastic trays and processed as described above After 2 ddead larvae were removed from trays and counted Also pupae were removed fromthe pupation medium on a daily basis

Basic biological and demographic parameters

To determine the demographic parameters of O bellus four groups with five pairsof newly emerged (lt24 h old) adult parasitoids in each group were placedindividually in cubical plexiglass cages (20 cm) and provided daily with ca A totalof 100 laboratory-reared 7ndash9 d-old A fraterculus larvae inside an oviposition unitplaced on the floor of the cage Water and honey were provided to the parasitoidsevery other day After a 4-h exposure to parasitoid females host larvae wereremoved and placed into plastic cups (8 cm diameter 5 cm height) with sterilisedvermiculite Pupae were held in these cups for five weeks to allow for adultemergence Emerged adult flies and parasitoids were taken out of each cup everyday counted and sexed The remaining non-eclosed puparia were dissected to checkfor the presence of parasitoid cadavers Wasprsquos mortality was recorded and the deadwasps were removed from each test cage on daily basis The assay was continueduntil all of the parasitoids died The basic biological parameters such as the pre-oviposition and oviposition periods adult lifespan development time (from egg toadult) offspring sex ratios (measured as the female proportion) and daily parasitismand emergence rates were determined for the O bellus cohort Furthermorestandard life table parameters (Carey 1993) were determined from the daily recordsof mortality and birth (Vargas et al 2002) The daily proportion of survivingparental females (lx) and the daily female offspring produced per female (mx) wereestimated and illustrated From the life table the following demographic parameterswere calculated according to formulae used by Carey (1993) gross fecundity rate netfecundity rate gross reproductive rate net reproductive rate intrinsic rate ofincrease finite rate of increase and mean generation time

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Data analysis

Parasitism was estimated as the total number of emerged and non-emergedparasitoids divided by the total number of obtained pupae and multiplied by 100whereas parasitoid emergence was calculated as the total number of emergedoffspring divided by the total number of recovered pupae and multiplied by 100Host mortality was estimated as the total number of dead larvae plus the totalnumber of puparia that did not yield insects divided by the total number of exposedhost larvae and multiplied by 100 The data regarding parasitoid emergenceparasitoid female offspring proportion (sex ratio) and host mortality were comparedthrough univariate General Linear Models (GLM) with a significance threshold ofP = 005 (Zar 1999) Mean comparisons were analysed by Tukeyrsquos honestysignificant difference (HSD) test at P = 005 Host mortality recorded from eachhost larval age trial was compared with its respective control through a t-test atP = 005 All of the proportional data were arcsine square root-transformed prior toanalysis Only the untransformed means (plusmn SE) are presented in the text

Results

A significant effect of host larval development stage on each response variable wasfound (F(415) = 42843 P lt 00001 for parasitoid emergence F(415) = 26782P lt 00001 for sex ratio F(415) = 10684 P lt 00001 for host mortality) Third-instarlarvae were most parasitised and produced a higher percentage of emerged O bellusand female offspring than those of the first and second instars (Figure 1) L3E and

Figure 1 Influence of A fraterculus instars on the percentages (mean plusmn SE) of emergedO bellus adults parasitoid female offspring (sex ratio) and host larval and pupal mortalityrecorded in optimal host larval-age tests Bars followed by the same letter indicate nosignificant differences (Tukey HSD test P = 005) Notations L1 first instars L2 secondinstars L3E early third-instars L3M middle third-instars L3L late third-instars

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L3M had the highest adult emergence values while both larval instars andL3L yielded statistically similar sex ratios (Figure 1) Among the third-instar larvaethe lowest host mortality percentage was recorded on L3E but was comparable tothat recorded from L1 (Figure 1) Host mortality recorded from L1 L2 L3E L3M andL3L was 25- 39- 49- 42- and 71-fold significantly higher than mortality recordedfrom their respective controls (t = 601 t = 942 t = 107 t = 583 t =114 for L1 L2L3E L3M and L3L respectively df = 18 and P lt 00001 for all five host age classes)

Both exposure time and ratio of hosts to parasitoids and their interaction had asignificant effect on parasitoid emergence and host mortality but not on the sex ratioof parasitoid (Table 1) Parasitoid emergence significantly increased at 41 61 81and 101 hostparasitoid ratios (Figure 2) and over the longer host larvae exposuretimes (4 and 5 h) (Figure 3) Percentage of parasitoid female offspring wassignificantly similar among all treatments and was generally male-biased Only at41 hostparasitoid ratio (Figure 2) and at 4 h exposure time (Figure 3) did O bellusexhibit a relatively female-biased sex ratio (1041 femalesmale) Host mortality waslower at a 21 hostparasitoid ratio (Figure 2) and over the shorter host exposuretimes (1ndash3 h) (Figure 3)

The pre-imaginal development times for male and female O bellus were 201 plusmn14 (mean plusmn standard error) and 216 plusmn 02 days at 25degC respectively A short pre-oviposition period (20 plusmn 06 days) was observed for mated females which producedeggs for 295 plusmn 14 days on average The mean female longevity was 303 plusmn 24 dayswhereas the mean male longevity was 274 plusmn 14 days At 32 days of age 50 of thefemales were still alive but at 60 d-old less than 10 of the females remained alive(Figure 4) The daily production of female offspring per parental female increaseduntil day 23 of the female reproductive life (23 plusmn 04 daughters per female) anddecreased thereafter (Figure 4)

The lifetime production of offspring (males plus females) of an average parentfemale was 207 plusmn 42 and the mean sex ratio (proportion of females) of the progenywas 580 plusmn 03 The net fecundity rate was 161 plusmn 67 offspring per newborn femaleThe mean number of female offspring produced by a single female during her

Table 1 Summary of univariate GLMs on the effect of host exposure time ratio of hosts toparasitoids and interactions of these categorical factors on parasitoid emergence parasitoidprogeny sex ratio and host mortality

Response variables

Parasitoidemergence

Parasitoidoffspring sex

ratio Host mortality

Source of variation df Error df F P F P F P

Hostparasitoidratio (HPR)

4 100 992 lt 00001a 038 = 08186 1525 lt 00001a

Host exposuretime (HET)

4 100 9154 lt 00001a 096 = 04319 276 = 00319a

HPR times HET 16 100 210 = 00137a 063 = 08454 440 lt 00001a

aSignificant variables

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

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(host larvae not exposed to parasitoids) were conducted for each larval age range todetermine the natural A fraterculus mortality rate Each test (including eachcontrol) was replicated 10 times Prior to the beginning of each replicate thequality of the A fraterculus larvae was assessed All the batches of host larvae withfly emergence percentages lt 90 were discarded and were not used in theexperiments

Optimal exposure time and ratio of hosts to parasitoids

To determine the optimal proportion of A fraterculus larvae per O bellus femaleand optimal amount of time to expose host larvae to parasitoids for rearing fivehostparasitoid ratios were individually analysed 21 41 61 81 and 101 Fivemated 10 d-old O bellus females that had prior oviposition experience and from acolony that was 14 generations old were used for each hostparasitoid ratiotreatment Each oviposition unit was filled with 7ndash9 d-old host larvae plus freshlarval diet Hosts at each density were exposed to parasitoid females in a separatecubical Plexiglas cages (20 cm) for 1 2 3 4 and 5 h After exposure to parasitoidslarvae were transferred into plastic trays and processed as described above After 2 ddead larvae were removed from trays and counted Also pupae were removed fromthe pupation medium on a daily basis

Basic biological and demographic parameters

To determine the demographic parameters of O bellus four groups with five pairsof newly emerged (lt24 h old) adult parasitoids in each group were placedindividually in cubical plexiglass cages (20 cm) and provided daily with ca A totalof 100 laboratory-reared 7ndash9 d-old A fraterculus larvae inside an oviposition unitplaced on the floor of the cage Water and honey were provided to the parasitoidsevery other day After a 4-h exposure to parasitoid females host larvae wereremoved and placed into plastic cups (8 cm diameter 5 cm height) with sterilisedvermiculite Pupae were held in these cups for five weeks to allow for adultemergence Emerged adult flies and parasitoids were taken out of each cup everyday counted and sexed The remaining non-eclosed puparia were dissected to checkfor the presence of parasitoid cadavers Wasprsquos mortality was recorded and the deadwasps were removed from each test cage on daily basis The assay was continueduntil all of the parasitoids died The basic biological parameters such as the pre-oviposition and oviposition periods adult lifespan development time (from egg toadult) offspring sex ratios (measured as the female proportion) and daily parasitismand emergence rates were determined for the O bellus cohort Furthermorestandard life table parameters (Carey 1993) were determined from the daily recordsof mortality and birth (Vargas et al 2002) The daily proportion of survivingparental females (lx) and the daily female offspring produced per female (mx) wereestimated and illustrated From the life table the following demographic parameterswere calculated according to formulae used by Carey (1993) gross fecundity rate netfecundity rate gross reproductive rate net reproductive rate intrinsic rate ofincrease finite rate of increase and mean generation time

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Data analysis

Parasitism was estimated as the total number of emerged and non-emergedparasitoids divided by the total number of obtained pupae and multiplied by 100whereas parasitoid emergence was calculated as the total number of emergedoffspring divided by the total number of recovered pupae and multiplied by 100Host mortality was estimated as the total number of dead larvae plus the totalnumber of puparia that did not yield insects divided by the total number of exposedhost larvae and multiplied by 100 The data regarding parasitoid emergenceparasitoid female offspring proportion (sex ratio) and host mortality were comparedthrough univariate General Linear Models (GLM) with a significance threshold ofP = 005 (Zar 1999) Mean comparisons were analysed by Tukeyrsquos honestysignificant difference (HSD) test at P = 005 Host mortality recorded from eachhost larval age trial was compared with its respective control through a t-test atP = 005 All of the proportional data were arcsine square root-transformed prior toanalysis Only the untransformed means (plusmn SE) are presented in the text

Results

A significant effect of host larval development stage on each response variable wasfound (F(415) = 42843 P lt 00001 for parasitoid emergence F(415) = 26782P lt 00001 for sex ratio F(415) = 10684 P lt 00001 for host mortality) Third-instarlarvae were most parasitised and produced a higher percentage of emerged O bellusand female offspring than those of the first and second instars (Figure 1) L3E and

Figure 1 Influence of A fraterculus instars on the percentages (mean plusmn SE) of emergedO bellus adults parasitoid female offspring (sex ratio) and host larval and pupal mortalityrecorded in optimal host larval-age tests Bars followed by the same letter indicate nosignificant differences (Tukey HSD test P = 005) Notations L1 first instars L2 secondinstars L3E early third-instars L3M middle third-instars L3L late third-instars

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L3M had the highest adult emergence values while both larval instars andL3L yielded statistically similar sex ratios (Figure 1) Among the third-instar larvaethe lowest host mortality percentage was recorded on L3E but was comparable tothat recorded from L1 (Figure 1) Host mortality recorded from L1 L2 L3E L3M andL3L was 25- 39- 49- 42- and 71-fold significantly higher than mortality recordedfrom their respective controls (t = 601 t = 942 t = 107 t = 583 t =114 for L1 L2L3E L3M and L3L respectively df = 18 and P lt 00001 for all five host age classes)

Both exposure time and ratio of hosts to parasitoids and their interaction had asignificant effect on parasitoid emergence and host mortality but not on the sex ratioof parasitoid (Table 1) Parasitoid emergence significantly increased at 41 61 81and 101 hostparasitoid ratios (Figure 2) and over the longer host larvae exposuretimes (4 and 5 h) (Figure 3) Percentage of parasitoid female offspring wassignificantly similar among all treatments and was generally male-biased Only at41 hostparasitoid ratio (Figure 2) and at 4 h exposure time (Figure 3) did O bellusexhibit a relatively female-biased sex ratio (1041 femalesmale) Host mortality waslower at a 21 hostparasitoid ratio (Figure 2) and over the shorter host exposuretimes (1ndash3 h) (Figure 3)

The pre-imaginal development times for male and female O bellus were 201 plusmn14 (mean plusmn standard error) and 216 plusmn 02 days at 25degC respectively A short pre-oviposition period (20 plusmn 06 days) was observed for mated females which producedeggs for 295 plusmn 14 days on average The mean female longevity was 303 plusmn 24 dayswhereas the mean male longevity was 274 plusmn 14 days At 32 days of age 50 of thefemales were still alive but at 60 d-old less than 10 of the females remained alive(Figure 4) The daily production of female offspring per parental female increaseduntil day 23 of the female reproductive life (23 plusmn 04 daughters per female) anddecreased thereafter (Figure 4)

The lifetime production of offspring (males plus females) of an average parentfemale was 207 plusmn 42 and the mean sex ratio (proportion of females) of the progenywas 580 plusmn 03 The net fecundity rate was 161 plusmn 67 offspring per newborn femaleThe mean number of female offspring produced by a single female during her

Table 1 Summary of univariate GLMs on the effect of host exposure time ratio of hosts toparasitoids and interactions of these categorical factors on parasitoid emergence parasitoidprogeny sex ratio and host mortality

Response variables

Parasitoidemergence

Parasitoidoffspring sex

ratio Host mortality

Source of variation df Error df F P F P F P

Hostparasitoidratio (HPR)

4 100 992 lt 00001a 038 = 08186 1525 lt 00001a

Host exposuretime (HET)

4 100 9154 lt 00001a 096 = 04319 276 = 00319a

HPR times HET 16 100 210 = 00137a 063 = 08454 440 lt 00001a

aSignificant variables

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

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Data analysis

Parasitism was estimated as the total number of emerged and non-emergedparasitoids divided by the total number of obtained pupae and multiplied by 100whereas parasitoid emergence was calculated as the total number of emergedoffspring divided by the total number of recovered pupae and multiplied by 100Host mortality was estimated as the total number of dead larvae plus the totalnumber of puparia that did not yield insects divided by the total number of exposedhost larvae and multiplied by 100 The data regarding parasitoid emergenceparasitoid female offspring proportion (sex ratio) and host mortality were comparedthrough univariate General Linear Models (GLM) with a significance threshold ofP = 005 (Zar 1999) Mean comparisons were analysed by Tukeyrsquos honestysignificant difference (HSD) test at P = 005 Host mortality recorded from eachhost larval age trial was compared with its respective control through a t-test atP = 005 All of the proportional data were arcsine square root-transformed prior toanalysis Only the untransformed means (plusmn SE) are presented in the text

Results

A significant effect of host larval development stage on each response variable wasfound (F(415) = 42843 P lt 00001 for parasitoid emergence F(415) = 26782P lt 00001 for sex ratio F(415) = 10684 P lt 00001 for host mortality) Third-instarlarvae were most parasitised and produced a higher percentage of emerged O bellusand female offspring than those of the first and second instars (Figure 1) L3E and

Figure 1 Influence of A fraterculus instars on the percentages (mean plusmn SE) of emergedO bellus adults parasitoid female offspring (sex ratio) and host larval and pupal mortalityrecorded in optimal host larval-age tests Bars followed by the same letter indicate nosignificant differences (Tukey HSD test P = 005) Notations L1 first instars L2 secondinstars L3E early third-instars L3M middle third-instars L3L late third-instars

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L3M had the highest adult emergence values while both larval instars andL3L yielded statistically similar sex ratios (Figure 1) Among the third-instar larvaethe lowest host mortality percentage was recorded on L3E but was comparable tothat recorded from L1 (Figure 1) Host mortality recorded from L1 L2 L3E L3M andL3L was 25- 39- 49- 42- and 71-fold significantly higher than mortality recordedfrom their respective controls (t = 601 t = 942 t = 107 t = 583 t =114 for L1 L2L3E L3M and L3L respectively df = 18 and P lt 00001 for all five host age classes)

Both exposure time and ratio of hosts to parasitoids and their interaction had asignificant effect on parasitoid emergence and host mortality but not on the sex ratioof parasitoid (Table 1) Parasitoid emergence significantly increased at 41 61 81and 101 hostparasitoid ratios (Figure 2) and over the longer host larvae exposuretimes (4 and 5 h) (Figure 3) Percentage of parasitoid female offspring wassignificantly similar among all treatments and was generally male-biased Only at41 hostparasitoid ratio (Figure 2) and at 4 h exposure time (Figure 3) did O bellusexhibit a relatively female-biased sex ratio (1041 femalesmale) Host mortality waslower at a 21 hostparasitoid ratio (Figure 2) and over the shorter host exposuretimes (1ndash3 h) (Figure 3)

The pre-imaginal development times for male and female O bellus were 201 plusmn14 (mean plusmn standard error) and 216 plusmn 02 days at 25degC respectively A short pre-oviposition period (20 plusmn 06 days) was observed for mated females which producedeggs for 295 plusmn 14 days on average The mean female longevity was 303 plusmn 24 dayswhereas the mean male longevity was 274 plusmn 14 days At 32 days of age 50 of thefemales were still alive but at 60 d-old less than 10 of the females remained alive(Figure 4) The daily production of female offspring per parental female increaseduntil day 23 of the female reproductive life (23 plusmn 04 daughters per female) anddecreased thereafter (Figure 4)

The lifetime production of offspring (males plus females) of an average parentfemale was 207 plusmn 42 and the mean sex ratio (proportion of females) of the progenywas 580 plusmn 03 The net fecundity rate was 161 plusmn 67 offspring per newborn femaleThe mean number of female offspring produced by a single female during her

Table 1 Summary of univariate GLMs on the effect of host exposure time ratio of hosts toparasitoids and interactions of these categorical factors on parasitoid emergence parasitoidprogeny sex ratio and host mortality

Response variables

Parasitoidemergence

Parasitoidoffspring sex

ratio Host mortality

Source of variation df Error df F P F P F P

Hostparasitoidratio (HPR)

4 100 992 lt 00001a 038 = 08186 1525 lt 00001a

Host exposuretime (HET)

4 100 9154 lt 00001a 096 = 04319 276 = 00319a

HPR times HET 16 100 210 = 00137a 063 = 08454 440 lt 00001a

aSignificant variables

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

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L3M had the highest adult emergence values while both larval instars andL3L yielded statistically similar sex ratios (Figure 1) Among the third-instar larvaethe lowest host mortality percentage was recorded on L3E but was comparable tothat recorded from L1 (Figure 1) Host mortality recorded from L1 L2 L3E L3M andL3L was 25- 39- 49- 42- and 71-fold significantly higher than mortality recordedfrom their respective controls (t = 601 t = 942 t = 107 t = 583 t =114 for L1 L2L3E L3M and L3L respectively df = 18 and P lt 00001 for all five host age classes)

Both exposure time and ratio of hosts to parasitoids and their interaction had asignificant effect on parasitoid emergence and host mortality but not on the sex ratioof parasitoid (Table 1) Parasitoid emergence significantly increased at 41 61 81and 101 hostparasitoid ratios (Figure 2) and over the longer host larvae exposuretimes (4 and 5 h) (Figure 3) Percentage of parasitoid female offspring wassignificantly similar among all treatments and was generally male-biased Only at41 hostparasitoid ratio (Figure 2) and at 4 h exposure time (Figure 3) did O bellusexhibit a relatively female-biased sex ratio (1041 femalesmale) Host mortality waslower at a 21 hostparasitoid ratio (Figure 2) and over the shorter host exposuretimes (1ndash3 h) (Figure 3)

The pre-imaginal development times for male and female O bellus were 201 plusmn14 (mean plusmn standard error) and 216 plusmn 02 days at 25degC respectively A short pre-oviposition period (20 plusmn 06 days) was observed for mated females which producedeggs for 295 plusmn 14 days on average The mean female longevity was 303 plusmn 24 dayswhereas the mean male longevity was 274 plusmn 14 days At 32 days of age 50 of thefemales were still alive but at 60 d-old less than 10 of the females remained alive(Figure 4) The daily production of female offspring per parental female increaseduntil day 23 of the female reproductive life (23 plusmn 04 daughters per female) anddecreased thereafter (Figure 4)

The lifetime production of offspring (males plus females) of an average parentfemale was 207 plusmn 42 and the mean sex ratio (proportion of females) of the progenywas 580 plusmn 03 The net fecundity rate was 161 plusmn 67 offspring per newborn femaleThe mean number of female offspring produced by a single female during her

Table 1 Summary of univariate GLMs on the effect of host exposure time ratio of hosts toparasitoids and interactions of these categorical factors on parasitoid emergence parasitoidprogeny sex ratio and host mortality

Response variables

Parasitoidemergence

Parasitoidoffspring sex

ratio Host mortality

Source of variation df Error df F P F P F P

Hostparasitoidratio (HPR)

4 100 992 lt 00001a 038 = 08186 1525 lt 00001a

Host exposuretime (HET)

4 100 9154 lt 00001a 096 = 04319 276 = 00319a

HPR times HET 16 100 210 = 00137a 063 = 08454 440 lt 00001a

aSignificant variables

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

Biocontrol Science and Technology 385

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

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lifetime was 120 plusmn 29 The net reproductive rate was 96 plusmn 25 females per newbornfemale and the mean generation time was 84 plusmn 02 days The intrinsic rate ofincrease was 006 plusmn 001 femalefemale per day and the increase in finite rate was106 plusmn 001 per day

Mean daily parasitism was lower than 1 when females were 0ndash3 d-old butgradually increased until the females were 4ndash5 d-old then ranged between 10 and15 until the females were 22ndash23 d-old and decreased thereafter (Figure 5) Thecumulative lifetime number of larvae parasitised per female was 279 plusmn 06 whereas

Figure 2 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different hostparasitoid ratios Bars followed by thesame letter indicate no significant differences (Tukey HSD test P = 005)

Figure 3 Mean (plusmn SE) percentage of O bellus emergence sex ratio of parasitoid offspring(per cent females) and host mortality (percentage of dead host larvae plus non-eclosed hostpupae) recorded from A fraterculus at different host exposure times to parasitoid femalesBars followed by the same letter indicate no significant differences (Tukey HSD testP = 005)

382 P Schliserman et al

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each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

384 P Schliserman et al

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ded

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New

cast

le U

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11

41 0

8 M

ay 2

014

attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

Biocontrol Science and Technology 385

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] at

11

41 0

8 M

ay 2

014

Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

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] at

11

41 0

8 M

ay 2

014

each day 46 plusmn 01 larvae were parasitised by a female Per cent offspring emergencewas highest among the 20 and 24 d-old females and decreased for older females(Figure 5) The mean daily rate of emerged adults was 27 plusmn 11

Figure 4 Daily survival (lx) (black circle) and daily female offspring produced per female (mx)(white circle) of O bellus (14 generations old) reared on 7- to 9-d-old (early and middle third-instars) A fraterculus larvae

Figure 5 Mean (plusmn SD) parasitism and emergence percentages of an age range of O bellusfemales (14 generations old) reared on 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae

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Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

384 P Schliserman et al

Dow

nloa

ded

by [

New

cast

le U

nive

rsity

] at

11

41 0

8 M

ay 2

014

attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

Biocontrol Science and Technology 385

Dow

nloa

ded

by [

New

cast

le U

nive

rsity

] at

11

41 0

8 M

ay 2

014

Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

Dow

nloa

ded

by [

New

cast

le U

nive

rsity

] at

11

41 0

8 M

ay 2

014

Discussion

Suitable host age and high host quality as well as adequate handling procedures infruit fly prepupal-larval parasitoid rearing are needed to achieve efficient adultproduction (Wong amp Ramadan 1992) Both host exposure time and ratio of hostlarvae to parasitoids correlated with the parental female age (Ramadan Wong ampBeardsley 1989) are particularly useful for this purpose Thus both parasitoidemergence and progeny sex ratio significantly influence the success rate of theparasitoid rearing process (Aluja et al 2009 Cancino Ruiz Sivinski Gaacutelvez ampAluja 2009) In this regard 7ndash9 d-old (early and middle third-instars) A fraterculuslarvae at a low ratio (41) of host to parasitoid were exposed to 10 d-old O bellusfemales for a relatively long time (4 h) this array of factors was sufficient to achievethe highest average adult emergence (48) and an offspring sex ratio at equitableproportion Neither increasing hostparasitoid ratio over 41 nor exposing the host toa time beyond 4 h significantly enhanced overall parasitoid female offspring yield

Overall the biological parameter values estimated for O bellus are within thegeneral range of data recorded for other neotropical opiine fruit fly parasitoid speciesby Aluja et al 2009 (Table 2) However O bellus exhibited various interesting

Table 2 Handling procedures for rearing four neotropical Anastrepha larval-prepupal opiineparasitoid species and summary of their biological parameters described by Aluja et al 2009

Rearing handling procedures Parasitoid species

Biological parametersDoryctobracon

areolatusaDoryctobraconcrawfordib

Utetesanastrephaec

Opiushirtusa

Ndeg parasitoid females perexperimental cage

30 30 40 40

Host exposure periods (h) 36 7ndash36 7ndash48 7ndash36Exposed hosts per parasitoidfemale

18ndash831 18ndash191 14ndash641 14ndash641

Parasitoid emergence () 11ndash24 21ndash37 20ndash26 14ndash25offspring sex ratio(female male)

08ndash121 13ndash151 08ndash141 08ndash131

GFR (gross fecundity rateoffspringparent female)

661 plusmn 175 2915 plusmn 830 287 plusmn 040 627 plusmn 204

NFR (net fecundity rateoffspringnewborn female)

219 plusmn 041 1068 plusmn 140 264 plusmn 034 214 plusmn 037

Ro (net reproductive ratefemale offspringnewbornfemale)

139 plusmn 016 536 plusmn 066 134 plusmn 020 127 plusmn 013

r (intrinsic rate ofincrease day)

003 plusmn 001 024 plusmn 004 007 plusmn 004 003 plusmn 001

λ (finite rate of increaseper day)

104 plusmn 001 127 plusmn 005 108 plusmn 004 103 plusmn 001

T (mean generationtime days)

865 plusmn 087 769 plusmn 144 308 plusmn 039 846 plusmn 068

The host the rearing experimental conditions and the generation were aAnastrepha ludens Tdeg 25 plusmn 1degCRH 70 plusmn 5 and F14

bA ludens Tdeg 23 plusmn 2degC RH 70 plusmn 5 and F14cA ludens Tdeg 25 plusmn 1degC RH 70 plusmn

5 and F9

384 P Schliserman et al

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

Biocontrol Science and Technology 385

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

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attributes For example (1) average parasitoid emergence rate was clearly 15- to4-fold higher than values shown in Table 2 (2) female longevity was almost 3-foldhigher than that of Doryctobracon crawfordi (Viereck) D areolatus (Szeacutepligeti)Utetes anastrephae (Viereck) and Opius hirtus (Fischer) (Braconidae Opiinae) and(3) the net reproductive rate value was notably 2- to 7-fold higher than thoserecorded for all four native opiine species in Table 2 the intrinsic rate of increasevalue was 2-fold higher than that of D areolatus and O hirtus and the meangeneration time value was closer to those found for D crawfordi D areolatus andO hirtus but it was about 3-fold higher than that recorded for U anastrephae Incontrast net reproductive rate intrinsic rate of increase and mean generation timevalues recorded here for O bellus were 2- to 5-fold lower than those described byVargas et al (2002) and Viscarret La Rossa Segura Ovruski and Cladera (2006)for the exotic opiine D longicaudata reared on Bactrocera dorsalis (Hendel) and onC capitata respectively However ovipositing O bellus females had a meanlongevity comparable to that reported by Vargas et al (2002) Viscarret et al(2006) and Gonzaacutelez Montoya Peacuterez-Lachaud Cancino and Liedo (2007) forD longicaudata Rearing procedures and handling conditions such as parasitoidfemalecage density host exposure period and hostparasitoid ratio host species usedfor rearing (Cancino amp Montoya 2004) or a different adaptability to laboratoryconditions for each parasitoid species (Eitam et al 2003) could explain differences inparameter values However the three findings highlighted above for O bellus addedto the relatively fast adaptation of this native parasitoid species to laboratoryconditions and are particularly important they indicate that their rearing mightincrease at a greater rate thereby facilitating parasitoid production Similarobservations were also reported by Aluja et al 2009 for D crawfordi whose mass-rearing has already taken place successfully in the fruit fly and parasitoid facility ofthe Medfly-Moscafrut programme in Metapa de Domiacutenguez Chiapas Mexico(Cancino et al 2009)

The results of the present study provide for the first time information on theperformance of O bellus reared on A fraterculus larvae under laboratory conditionsThese results may be used to develop a more efficient rearing method for thisneotropical opine species for biological control purposes in Argentina howeverunderstanding all the factors that influence both offspring production and offspringsex ratio is critical for successful parasitoid rearing Therefore additional studies inthe laboratory focusing on factors that influence offspring sex ratios (Heimpel ampLundgren 2000 Montoya Cancino Peacuterez-Lachaud amp Liedo 2011) may be neededto achieve cost-effective production in the O bellus rearing process Nevertheless thelong lifespan and the reproductive parameter values estimated here suggest that thisparasitoid species has suitable attributes for mass-rearing and augmentative releasesFinally the biological information provided here can be used to pursue comparativestudies of native parasitoid reproduction and its relationship to environment andhost-range

AcknowledgementsWe are grateful to Patricia Colombres Omar Ulises Chaya Guillermo Borchia LilianaColombres Carolina Chiappini Natalia Salinas and Emilia Rodriguez (PROIMI) forvaluable technical assistance Special thanks go to Juan Rull for revision and suggestions onimproving the manuscript to Luisa Montivero for providing language assistance and to

Biocontrol Science and Technology 385

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] at

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Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

Dow

nloa

ded

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New

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le U

nive

rsity

] at

11

41 0

8 M

ay 2

014

Segundo Nuacutentildeez-Campero (PROIMI) for his advice during database organisation We alsothank two anonymous reviewers and the editor for helping us produce a better articleFinancial support was provided by the Consejo Nacional de Investigaciones Cientiacuteficas yTeacutecnicas de la Repuacuteblica Argentina (CONICET) (grant PIP2008 No 112-200801-01353) andby the Agencia Nacional de Promocioacuten Cientiacutefica y Tecnoloacutegica de Argentina (ANCyT)through the Fondo Nacional de Ciencia y Tecnologiacutea (FONCyT) (grants PICT2006 No 2402and PICT2010 No 0393)

ReferencesAguiar-Menezes E L amp Menezes E B (2001) Parasitismo Sazonal e Flutuaccedilatildeo

Populacional de Opiinae (Hymenoptera Braconidae) Parasitoacuteides de Espeacutecies de Anastre-pha (Diptera Tephritidae) em Seropeacutedica RJ [Seasonal parasitism and populationfluctuation of Opiinae (Hymenoptera Braconidae) parasitoids of Anastrepha species inSeropedica RJ] Neotropical Entomology 30 613ndash623 doi101590S1519-566X2001000400016

Aluja M amp Rull J (2009) Managing pestiferous fruit flies (Diptera Tephritidae) throughenvironmental manipulation In M Aluja TC Leskey amp C Vincent (Eds) Biorationaltree fruit pest management (pp 171ndash213) Oxfordshire CABI

Aluja M Sivinski J Ovruski S M Guilleacuten L Loacutepez M Cancino J hellip Ruiz L (2009)Colonization and domestication of seven species of native new world HymenopterousLarval-Prepupal and Pupal fruit fly (Diptera Tephritidae) Parasitoids Biocontrol Science ampTechnology 19 49ndash79 doi10108009583150802377373

Canal N A amp Zucchi R A (2000) Parasitoacuteides-braconidae [Parasitoids-braconidae] In AMalavasi amp R A Zucchi (Eds) Moscas-das-frutas de Importacircncia Econocircmica no BrasilConhecimento Baacutesico e Aplicado ( pp 119ndash126) Riberatildeo Preto Holos Editora

Cancino J amp Montoya P (2004) Desirable attributes of mass reared parasitoids for fruit flycontrol A comment Vedalia (Meacutexico) 11 53ndash58

Cancino J Ruiz L Sivinski J Gaacutelvez F O amp Aluja M (2009) Rearing of fivehymenopterous larval-prepupal (Braconidae Figitidae) and three Pupal (DiapriidaeChalcididae Eurytomidae) native parasitoids of the genus Anastrepha (Diacuteptera Tephriti-dae) on irradiated A ludens larvae and pupae Biocontrol Science amp Technology 19 193ndash209 doi10108009583150802377423

Carey J R (1993) Applied demography for biologists with special emphasis on insects NewYork NY Oxford University Press

De Jesuacutes C R Pereira J D B De Oliveira M N Da Silva R A Souza-Filho M FDa Costa-Neto S V hellip Zucchi R A (2008) New records of fruit flies (DipteraTephritidae) wild hosts and parasitoids (Hymenoptera Braconidae) in the BrazilianAmazon Neotropical Entomology 37 733ndash734 doi101590S1519-566X2008000600017

Eitam A Holler T Sivinski J amp Aluja M (2003) Use of host fruit chemical cues forlaboratory rearing of Doryctobracon areolatus (Hymenoptera Braconidae) a parasitoid ofAnastrepha spp (Diptera Tephritidae) Florida Entomologist 86 211ndash216 doi1016530015-4040(2003)086[0211UOHFCC]20CO2

Friacuteas D Selivon D amp Hernaacutendez-Ortiz V (2008) Taxonomy of immature stages Newmorphological characters for Tephritidae larvae identification In R Sugayama R AZucchi S Ovruski and J Sivinski (Eds) Fruit flies of economic importance from basic toapplied knowledge (pp 29ndash44) Bahiacutea Programa Moscamed Brazil

Garciacutea-Medel D Sivinski J Diacuteaz-Fleischer F Ramirez-Romero R amp Aluja M (2007)Foraging behavior by six fruit fly parasitoids (Hymenoptera Braconidae) released as single-or multiple-species cohorts in field cages Influence of fruit location and host densityBiological Control 43 12ndash22 doi101016jbiocontrol200706008

Gates M W Heraty J M Schauff M E Wagner D L Whitfield J B amp Wahl D B(2002) Survey of the parasitic hymenoptera on Leafminers in California Journal ofHymenoptera Research 11 213ndash270

Gonzaacutelez P I Montoya P Peacuterez-Lachaud G Cancino J amp Liedo P (2007)Superparasitism in mass reared Diachasmimorpha longicaudata (Asmead) (Hymenoptera

386 P Schliserman et al

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Braconidae) a parasitoid of fruit flies (Diptera Tephritidae) Biological Control 40 320ndash326 doi101016jbiocontrol200611009

Guilleacuten D amp Saacutenchez R (2007) Expansion of the national fruit fly control programme inArgentina In M J B Vreysen A S Robinson amp J Hendrichs (Eds) Area-wide control ofinsects pests From research to field implementation (pp 653ndash660) Dordrecht Springer

Heimpel G E amp Lundgren J G (2000) Sex ratios of commercially reared biologicalcontrol agents Biological Control 19 77ndash93 doi101006bcon20000849

Hernaacutendez-Ortiz V Gonzaacutelez H D Escalante-Tio A amp Manrique-Saide P (2006)Hymenopteran parasitoids of Anastrepha fruit flies (Diptera Tephritidae) reared fromdifferent hosts in Yucatan Mexico Florida Entomologist 89 508ndash514 doi1016530015-4040(2006)89[508HPOAFF]20CO2

Jonsson M Wratten S D Landis D A amp Gurr G M (2008) Recent advances inconservation biological control of arthropods by arthropods Biological Control 45 172ndash175 doi101016jbiocontrol200801006

Katiyar K P Camacho J Geraud F amp Matheus R (1995) Parasitoides himenoacutepteros demoscas de las frutas (Diptera Tephritidae) en la regioacuten occidental de Venezuela[Hymenopterous parasitoids of fruit flies in western Venezuela] Revista de la Facultad deAgronomiacutea de Venezuela 12 303ndash312

Medianero E Korytkowski C A Campo C amp de Leoacuten C (2006) Hymenopteraparasitoides asociados a Anastrepha (Diptera Tephritidae) en Cerro Jefe y Altos de PacoraPanama [Hymenopterous parasitoids associated with Anastrepha in Cerro Jefe and Altos dePacora Panamaacute] Revista Colombiana de Entomolologiacutea 32 136ndash139

Montoya P Cancino J Peacuterez-Lachaud G amp Liedo P (2011) Host size superparasitismand sex ratio in mass-reared Diachasmimorpha longicaudata a fruit fly parasitoidBioControl 56 11ndash17 doi101007s10526-010-9307-9

Ovruski S M Aluja M Sivinski J amp Wharton R A (2000) Hymenopteran parasitoidson fruit-infesting Tephritidae (Diptera) in Latin America and the Southern United StatesDiversity distribution taxonomic status and their use in fruit fly biological controlIntegrated Pest Management Reviews 5 81ndash107 doi101023A1009652431251

Ovruski S M amp Schliserman P (2012) Biological control of Tefhritid fruit flies inArgentina Historical review current status and future trends for developing a parasitoidmass-release program Insects 3 1ndash19 doi103390insects3030870

Ovruski S M Schliserman P amp Aluja M (2004) Indigenous parasitoids (Hymenoptera)attacking Anastrepha fraterculus and Ceratitis capitata (Diptera Tephritidae) in native andexotic host plants in Northwestern Argentina Biological Control 29 43ndash57 doi101016S1049-9644(03)00127-0

Ovruski S M Schliserman P Nuntildeez-Campero S R Orontildeo L E Bezdjian L PAlbornoz-Medina P amp Van Nieuwenhove G A (2009) A survey of hymenopterouslarval-pupal parasitoids associated with Anastrepha fraterculus and Ceratitis capitata(Diptera Tephritidae) infesting wild guava (Psidium guajava) and peach (Prunus persica)in the Southernmost section of the Bolivian Yungas forest Florida Entomologist 92 269ndash275 doi1016530240920211

Ramadan M M Wong T T Y amp Beardsley J W (1989) Survivorship potencial andrealized fecundity of Biosteres tryoni (Hymenoptera Braconidae) A larval parasitoid ofCeratitis capitata (Diptera Tephritidae) Entomophaga 34 291ndash297 doi101007BF02372468

Sivinski J Aluja M amp Loacutepez M (1997) The spatial and temporal distributions ofparasitoids of mexican Anastrepha species (Diptera Tephritidae) within the canopies of fruittrees Annals of the Entomological Society of America 90 604ndash618

Sivinski J Pintildeero J amp Aluja M (2000) The distributions of parasitoids (Hymenoptera) ofAnastrepha fruit flies (Diptera Tephritidae) along an altitudinal gradient in VeracruzMexico Biological Control 18 258ndash269 doi101006bcon20000836

Sivinski J Vulinec K amp Aluja M (2001) Ovipositor length in a guild of parasitoids(Hymenoptera Braconidae) attacking Anastrepha spp fruit flies (Diptera Tephritidae) inSouthern Mexico Annals of the Entomological Society of America 94 886ndash895doi1016030013-8746(2001)094[0886OLIAGO]20CO2

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Uchocirca-Fernandes M A da Silva Molina R M de Oliveira I Zucchi R A Canal A ampDiacuteaz N B (2003) Larval endoparasitoids (Hymenoptera) of frugivorous flies (DipteraTephritoidea) reared from fruits of the Cerrado of the State of Mato Grosso do Sul BrazilRevista Brasilera de Entomologia 47 181ndash186 doi101590S0085-56262003000200005

Vargas R I Ramadan M Hussain T Mochizuki N Bautista R C amp Stark J D(2002) Comparative demography of six fruit fly (Diptera Tephritidae) parasitoids(Hymenoptera Braconidae) Biological Control 25 30ndash40 doi101016S1049-9644(02)00046-4

Viscarret M M La Rossa R Segura D F Ovruski S M ampCladera J L (2006)Evaluation of the parasitoid Diachasmimorpha longicaudata (Ashmead) (HymenopteraBraconidae) reared on a genetic sexing strain of Ceratitis capitata (Wied) (DipteraTephritidae) Biological Control 36 147ndash153 doi101016jbiocontrol200508009

Wharton R A (1997) Generic relationships of opiine braconidae (Hymenoptera) parasiticon fruit-infesting Tephritidae (Diptera) Contributions of the American Entomology Institute30 1ndash53

Wharton R A ampMarsh P M (1978) New world Opiinae (Hymenoptera Braconidae)parasitic on Tephritidae (Diptera) Journal of Washington Academy of Science 68 147ndash167

Wong T T Y amp Ramadan M M (1992) Mass rearing biology of larval parasitoids(Hymenoptera Braconidae Opiinae) of Tephritid flies (Diptera Tephritidae) in Hawaii InT E Anderson amp N C Leppla (Eds) Advances in insect rearing for research and pestmanagement (pp 405ndash426) San Francisco Westview Press

Zar J H (1999) Biostatistical analysis New Jersey Prentice Hall

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