AP QUEIROZ 1,AFBUENO 2,APOMARI-FERNANDES 3, MLM GRANDE 4,OCBORTOLOTTO 4,DMSILVA 1. Introduction BIOLOGICAL CONTROL

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1 Neotrop Entomol DOI /s BIOLOGICAL CONTROL Low Temperature Storage of Telenomus remus (Nixon) (Hymenoptera: Platygastridae) and its Factitious Host Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) AP QUEIROZ 1,AFBUENO 2,APOMARI-FERNANDES 3, MLM GRANDE 4,OCBORTOLOTTO 4,DMSILVA 1 1 Instituto Agronômico do Paraná (IAPAR), Londrina, Paraná, Brasil 2 Empresa Brasileira de Pesquisa Agropecuária Embrapa Soja, Londrina, Paraná, Brasil 3 Univ Federal da Fronteira Sul, Laranjeiras do Sul, Paraná, Brasil 4 Univ Estadual de Londrina, Londrina, Paraná, Brasil Keywords Mass rearing, biological control, parasitoid shelf life Correspondence AF Bueno, Empresa Brasileira de Pesquisa Agropecuária Embrapa Soja, Londrina, Paraná, Brasil ; adeney.bueno@embrapa.br Edited by Tiago C da Costa Lima Embrapa Received 16 February 2016 and accepted 29 August 2016 * Sociedade Entomológica do Brasil 2016 Abstract We conducted three bioassays to evaluate the effect of low-temperature storage of eggs (host) and pupae and adults (parasitoid) on the biology and parasitism capacity of the egg parasitoid Telenomus remus (Nixon) (Hymenoptera: Platygastridae). Viable stored Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) eggs were parasitized to the same degree or even higher than fresh eggs when stored until 14 days at 5 C or until 21 days at 10 C. In contrast, the percentage of parasitized sterilized eggs was equal to the control only when stored for 7 and 14 days. Survival of T. remus pupae declined with storage time at both studied temperatures (5 and 10 C). However, after 7 days of storage, survival of pupae was still 86.3 and 64.9% at 10 and 5 C, respectively. The number of adult male survivors remained similar until the fourth storage day at both 5 and 10 C. In contrast, female survival did not differ until day 8 at 10 C or day 6 at 5 C. Parasitism capacity of stored adults was not altered by storage compared with the control. Therefore, we conclude that the maximal storage time at 10 C is 21 days for viable C. cephalonica eggs and 7 days for T. remus pupae, while parasitoid adults should not be stored for more than 4 days at either 5 or 10 C. Introduction Telenomus remus (Nixon) (Hymenoptera: Platygastridae) is an effective egg parasitoid of pests in the genus Spodoptera (Pomari et al 2012), which includes species that are able to cause damage to economically important crops. Spodoptera species attack crops such as maize (Cruz et al 1999) and other grasses (Pogue 2002), eucalyptus (Santos et al 1980), and soybean (Sá et al 2009). The potential of T. remus as a biological control tool for genus Spodoptera is mainly due to its high reproductive capacity (Cave 2000; Bueno et al 2008). Each parasitoid female produces around 270 eggs during its lifespan, usually laid individually on each host egg (Cave 2000). Moreover, T. remus is especially noteworthy for its effective action on eggs of Spodoptera spp. in superposed layers. This species can parasitize eggs located in the inner layers of the egg mass (Bueno et al 2008),apropertyrarelyobservedwithothereggparasitoids. In addition, T. remus has high dispersal and host search capacities (Pomari et al 2013), underlining its potential for augmentative biological control programs. Despite these traits of T. remus that are desirable for biological control, rearing is only performed at small scales due to inherent costs and challenges (Pomari-Fernandes et al 2015). Successful large-scale rearing of parasitoids is essential for the implementation of augmentative biological control programs (Corrêa-Ferreira & Moscardi 1993). T. remus can be reared in eggs of a factitious host, the rice moth Corcyra

2 Queiroz et al cephalonica (Stainton) (Lepidoptera: Pyralidae) for largescale production (Pomari-Fernandes et al 2015). Maintenance throughout the year is essential for successful parasitoid rearing, because short interruptions can cause problems that hinder the return to the desired insect production level (Pratissoli et al 2003). Therefore, appropriate storage techniques are required for hosts and parasitoids in order to optimize the mass rearing needed to carry out biological control programs (López & Botto 2005). These techniques contribute to reducing insect production costs while producing the necessary number of parasitoids to be released in the field. Parasitoid production at low costs is crucial for the parasitoid to become an economically viable tool in biological control programs (Bernardo et al 2008). Additionally, storage techniques are essential to increase the insects shelf life, the time period during which they can be used for their intended purposes (Macedo et al 2006). An increase in shelf life is usually inversely proportional to production costs, which can be decisive for the success or failure of a biological control program (Macedo et al 2006). In order to evaluate different storage techniques, possible negative effects on insect quality must be taken into account (Colinet & Boivin 2011). Generally, the performance of stored hosts and their natural enemies depends on storage temperature and duration (Lysyk 2004). Thus, with respect to the rearing of T. remus, information on the influence of low-temperature storage on C. cephalonica eggs and on pupae and adults of T. remus is of great theoretical and practical interest. Data on the biological and reproductive parameters related to parasitism capacity (%), parasitoid emergence (%), and adult longevity (days) can help to evaluate the performance of the parasitoid after storage (Leopold 1998). Like all insect species (Leopold 1998), both T. remus pupae or adults and C. cephalonica eggs exhibit a level of cold tolerance before freezing or permanent injury (Gautam 1986). However, low temperature tolerance is a flexible trait determined by a broad range of factors (biotic and abiotic) experienced before, during, or after cold exposure (Colinet & Boivin 2011). Thus, our study aimed to evaluate the effects of low-temperature storage on C. cephalonica eggs and on T. remus pupae and adults under fixed conditions in order to optimize the mass rearing of this parasitoid in the laboratory. Material and methods This study was carried out in the laboratories of Embrapa Soybean, Londrina, State of Paraná, under controlled conditions (T = 25 ± 2 C; RH = 80 ± 10%; photoperiod = 14/10 h L/D) inside Biochemical Oxygen Demand (BOD) climate chambers (ELETROLab, model EL 212, São Paulo, SP, Brazil). The study consisted of three bioassays: (1) evaluation of C. cephalonica eggs exposed to UV light for 30 min and stored at temperatures of 5 and 10 C for zero (control), 7, 14, and 21 days; (2) evaluation of pupae of T. remus stored at 5 and 10 C for zero (control), 7, 14, and 21 days; and (3) evaluation of adults of T. remus stored at 5 and 10 C for zero (control), 2, 4, 6, and 8 days. After each storage period, all treatments were moved to BODs (ELETROLab, model EL 212, São Paulo, SP, Brazil) under controlled conditions (T = 25 ± 2 C; RH = 80 ± 10%; photoperiod = 14/10 h L/D). The C. cephalonica eggs used in this study were produced following the methodology of Bernardi et al (2000). T. remus pupae and adults were reared from C. cephalonica eggs over several successive generations following the methodology of Pomari-Fernandes et al (2015). Bioassay 1: Storage of Corcyra cephalonica eggs The experiment was carried out in a completely randomized design with 14 treatments (Table 1) and four replicates. First, the C. cephalonica eggs (up to 24 h) were collected and then sterilized according to established procedures. The eggs were placed in Petri dishes (5 cm 1 cm), covered with aluminum foilandstoredat5and10 CinsideBODchambers (ELETROLab, model EL 212, São Paulo, SP, Brazil) at 80 ± 10% RH, and a photoperiod of 14/10 h L/D. In order to assess any possible effect of low-temperature storage, C. cephalonica eggs stored for zero (control), 7, 14, and 21 days were exposed to parasitism by T. remus females (insects reared on C. cephalonica eggs F 50 generation) under controlled conditions (T = 25 ± 2 C; RH = 80 ± 10%; photoperiod = 14/10 h L/D). Recently, emerged females (up to 24 h) that had been previously mated and fed with honey were kept in Duran tubes (1.5 ml) throughout the experiment. Eggs of C. cephalonica were glued on cardboard cards (2.5 cm 5 cm) containing approximately 250 eggs. Each card was exposed to an individual female of T. remus in a Duran tube. Each replicate included an average of five tubes containing an individual female (four replicates five females/ replicate = 20 females/treatment). Parasitism was allowed to continue for 24 h. After that, the cards were kept in climate chambers with controlled temperature (25 ± 2 C), relative humidity (80 ± 10%), and photoperiod (14/10 h L/D) until the emergence of parasitoids. The number of parasitized eggs was recorded, along with parasitoid emergence (%) and sex ratio. Bioassay 2: Storage of Telenomus remus pupae The second experiment was carried out in a completely randomized design with nine treatments (Table 2) and five replicates. Cards with parasitized eggs at the pupa stage (10 days after parasitism) were stored at two different temperatures

3 Low Temperature Storage of Telenomus remus and Corcyra cephalonica Table 1 periods. Biological characteristics of Telenomus remus parasitoids in Corcyra cephalonica eggs stored at different temperatures and for different Treatment Number of parasitized eggs a Parasitoid emergence (%) Sex ratio Temperature of storage Egg type Days of storage viable none 11.3 ± 2.06cd 82.0 ± 1.79cde 0.6 ± 0.05ab sterilized none 10.2 ± 0.89d 93.3 ± 1.37ab 0.6 ± 0.06ab 5 C viable 7 days 23.7 ± 6.19bc 92.7 ± 1.08ab 0.7 ± 0.03ab 5 C viable 14 days 13.4 ± 2.39cd 90.0 ± 2.09abc 0.6 ± 0.04b 5 C viable 21 days 0.0 ± 0.00f 5 C sterilized 7 days 7.5 ± 0.59d 86.4 ± 1.39bcd 0.6 ± 0.05ab 5 C sterilized 14 days 0.60 ± 0.22ef 73.9 ± 3.89e 0.8 ± 0.11a 5 C sterilized 21 days 0.9 ± 0.56ef 77.5 ± 2.50de 0.5 ± 0.13b 10 C viable 7 days 37.1 ± 7.59b 94.4 ± 0.94ab 0.7 ± 0.04ab 10 C viable 14 days 67.3 ± 4.59a 97.2 ± 0.48a 0.6 ± 0.04ab 10 C viable 21 days 30.9 ± 3.71b 96.7 ± 0.85a 0.7 ± 0.03ab 10 C sterilized 7 days 31.8 ± 1.70b 91.5 ± 1.22ab 0.7 ± 0.02ab 10 C sterilized 14 days 4.6 ± 0.78de 87.5 ± 2.94bc 0.5 ± 0.11b 10 C sterilized 21 days 0.0 ± 0.00f - - CV (%) P < < F Means ± SEM followed by the same letter in a column do not differ statistically (Tukey test, P 0.05). - Parameter nonexistent. Control treatments (no stored eggs). ns ANOVA not significant. pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a Original results followed by statistical analysis performed on the transformed data into X þ 0:5. Table 2 Telenomus remus pupae survival after storage for different times at different temperatures. Treatment Temperature of storage Days of storage none 96.9 ± 0.46a 5 C 7 days 64.9 ± 2.22c 5 C 14 days 8.7 ± 1.01e 5 C 21 days 1.9 ± 0.58f 5 C 28 days 0.0 ± 0.00g 10 C 7 days 86.3 ± 1.09b 10 C 14 days 17.2 ± 1.82d 10 C 21 days 0.0 ± 0.00g 10 C 28 days 0.0 ± 0.00g CV (%) 7.3 P < F Parasitoid emergence (%) a Means ± SEM followed by the same letter in a column do not differ statistically (Tukey test, P 0.05). Control treatments (no stored pupae). a Original results followed by statistical analysis performed on the transformed data into arcsine of X=100. pffiffiffiffiffiffiffiffiffiffiffiffi (5 and 10 C) for periods of zero (control), 7, 14, 21, and 28 days to evaluate the storage impact on the parasitoid produced. For each replicate, approximately 500 parasitized C. cephalonica eggs containing the parasitoid pupae were placed in flat-bottomed glass tubes (8 cm 2 cm diameter) sealed with plastic wrap. The tubes were stored in BOD climate chambers at 5 and 10 C (ELETROLab, model EL 212, São Paulo, SP, Brazil, under 80 ± 10% RH and a photoperiod of 14/10 h L/D). Over four consecutive weeks, five replicates were evaluated at each temperature to assess the viability of the parasitoids at 7-day intervals. Along with the treatments, we stored 500 additional eggs at the same temperatures and time periods. This was done to evaluate the parasitism capacity of T. remus adults raised from stored pupae on eggs of its natural host Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) and in order to assess possible sublethal storage effects on the stored parasitoids. Using the natural host for this additional test was essential to simulate field conditions after parasitoid release. Parasitism capacity of T. remus adults from stored pupae was evaluated for newly emerged females (up to 24 h) which were individually placed in Duran tubes (1.5 ml) containing a droplet of honey as food. Egg masses with approximately 100 eggs of S. frugiperda were glued with non-toxic white glue (Tenaz ) onto labeled white cardboard cards (2.5 cm 5 cm).

4 Queiroz et al The cards were individually introduced into the tubes, and parasitism was allowed for 24 h. The experiment was performed in a BOD climate chamber set to a temperature of 25 ± 2 C, a relative humidity of 80 ± 10%, and a photoperiod of 14/10 h L/D. After parasitism, cards were removed and kept at the same conditions as during parasitism, until adults emerged. We evaluated the following T. remus biological parameters: number of parasitized eggs, duration of parasitoid egg-toadult period (days), parasitoid emergence (%), longevity of parental females (days), offspring sex ratio, and daily and cumulative parasitism. To determine the duration of the egg-to-adult period and the longevity of parental females, we made daily observations of emergence and death of adult T. remus. Bioassay 3: Storage of Telenomus remus adults The third experiment was conducted in a completely randomized design with eight treatments (Table 4) and ten replicates. For each replicate, five females and five newly emerged males (up to 24 h) were placed in individual glass tubes (8 cm 2 cm). The adult parasitoids were stored in BOD climate chambers at 5 and 10 C for periods of 2, 4, 6, and 8 days to evaluate the survival of the stored adults. Mortality was recorded in each storage period by counting dead and live individuals. At each evaluation day, a female T. remus of each replicate was separated to quantify parasitism capacity after different storage periods following the procedure for evaluating parasitism capacity described in bioassay 2. Statistical analysis The results of the three bioassays were analyzed by exploratory analyses to assess the assumptions of normality (Shapiro & Wilk 1965), variance homogeneity of the treatments (Burr & Foster 1972), and the additivity model for implementation of ANOVA. The results were compared by the Tukey test at a 5% error probability, using the statistical analysis program SAS (SAS Institute 2009). Results Bioassay 1: Storage of Corcyra cephalonica eggs The number of C. cephalonica eggs parasitized by T. remus after storage differed between viable and sterilized eggs. Those numbers (C. cephalonica eggs parasitized by T. remus after storage) were similar and even higher than the control (C. cephalonica eggs without storage) when viable C. cephalonica eggs were stored for until 14 days at 5 C or until 21 days at 10 C, respectively. In contrast, sterilized eggs were parasitized equally to the control only when stored until 7 days at 5 C or until 14 days at 10 C (Table 1). Importantly, the number of stored sterilized C. cephalonica eggs parasitized by T. remus was always similar or inferior to stored viable eggs at the same temperature and storage period (Table 1). The differences in storage conditions for C. cephalonica eggs also impacted the emergence of T. remus, with lower emergence when raised on sterilized C. cephalonica eggs stored at 5 C for 14 and 21 days. Differently, viable C. cephalonica eggs stored for 7 and 14 days at 5 C or for 7, 14, and 21 days at 10 C, and then offered to T. remus parasitism, had similar or even higher parasitoid emergence to the control (C. cephalonica eggs without storage) (Table 1). Overall, parasitism in cold storage treatments was similar to the control (over 80%), except for treatments that used sterilized C. cephalonica eggs stored at 5 C for 14 and 21 days (Table 1). Storage of C. cephalonica eggs did not alter T. remus sex ratio compared with the control, with all values being over 50% females (Table 1). Bioassay 2: Storage of Telenomus remus pupae Storage of T. remus pupae reduced their survival regardless of temperature or duration. All treatments resulted in pupal survival inferior to that observed for the control (96.9%) (Table 2). T. remus emergence (%) from stored pupae differed according to storage time and storage temperature (Table 2). The highest survival among stored pupae was recorded at 10 C for 7 days (86.3%) (Table 2). This was the only treatment with a survival rate higher than 80%. The parasitism capacity of adults from stored pupae also differed as a result of different storage conditions (Table 3). At the same storage temperature, the number of parasitized eggs, longevity of the parental females, and sex ratio were inversely proportional to an increase of storage time (Table 3). However, at 7 days of storage, all three parameters (number of parasitized eggs, parental longevity, and sex ratio) remained equal to the control at both 10 and 5 C (Table 3). Not only was T. remus emergence (%) from pupae stored at 10 C for 7 days the highest among treatments (Table 2), but the emerged adults also parasitized the highest number of S. frugiperda eggs compared to the other treatments, similar to T. remus emerged from pupae stored at 5 C for 7 days (Table 3). This treatment (stored pupae at 10 C for 7 days) did not differ from the control in almost all observed parameters (number of parasitized eggs, parasitism viability = parasitoid emergence, longevity of parasitoids, and sex ratio) (Table 3). Daily parasitism of T. remus (after storage of pupae) decreased throughout the lifespan of females at all temperatures and storage periods (Fig. 1). In the control treatment,

5 Low Temperature Storage of Telenomus remus and Corcyra cephalonica Table 3 Parasitism capacity of Telenomus remus adults from stored pupae in Spodoptera frugiperda eggs for different times at different temperatures. Treatment Temperature of storage Days of storage Number of parasitized eggs a Egg-to-adult period (days) Parasitoid emergence Parental female longevity (%) b (days) a Sex ratio none 49.8 ± 7.8a 12.5 ± 0.1a 88.3 ± 5.6 ns 4.3 ± 0.8a 0.6 ± 0.0a 5 C 7 days 50.3 ± 4.7a 13.3 ± 0.1c 89.6 ± ± 0.6a 0.6 ± 0.0a 5 C 14 days 8.4 ± 2.0b 13.0 ± 0.0b 89.9 ± ± 0.1b 0.2 ± 0.0bc 10 C 7 days 50.0 ± 0.3a 13.0 ± 0.0b 91.9 ± ± 0.2a 0.4 ± 0.0a 10 C 14 days 14.1 ± 1.8b 13.0 ± 0.0b 99.9 ± ± 0.1b 0.0 ± 0.0c CV (%) , P < < < F Means ± SEM followed by the same letter in a column do not differ statistically (Tukey test, P 0.05). Control treatments (no stored pupae). ns ANOVA not significant. p ffiffiffi a Original results followed by statistical analysis performed on the transformed data into X. pffiffiffiffiffiffiffiffiffiffiffiffi b Original results followed by statistical analysis performed on the transformed data into arcsine of X=100. the highest parasitism occurred within 24 h, resulting in an average of 40 parasitized eggs (Fig. 1a), and a cumulative parasitism level of 80% on the second day. After 7 days of storage at 5 C, the highest parasitism was observed within the first 24 h, with 30 parasitized eggs on average. The cumulative parasitism level of T. remus females was 80% on the second day only for adults from pupae stored for 7 days (Fig. 1b). At a storage temperature of 10 C, the highest parasitism was observed within the first 24 h by parasitoids grown from pupae stored for 7 and 14 days, with approximately 35 and 10 parasitized eggs, respectively (Fig. 1d and e). A cumulative parasitism level of 80% was observed on the second day only for females of T. remus stored for 7 days (Fig. 1d). Bioassay 3: Storage of Telenomus remus adults The survival of adult T. remus males and females was not impacted by storage of up to 4 days at both temperatures tested (Table 4). After 6 and 8 days of storage, mortality in males and females increased, respectively. Females were more storage tolerant as increased mortality was observed only for insects stored at 5 C for 8 days (Table 4). The parasitism capacity of parasitoids that survived storage as adults was not impacted by storage treatment. They parasitized a similar or even greater amount of S. frugiperda eggs compared with the control (adults without storage) (Table 5). All other biological characteristics evaluated (eggto-adult period, parasitoid emergence, parental female longevity, and sex ratio) also did not differ among treatments (Table 5). For all treatments, highest parasitism rates were observed within the first 24 h (Figs. 2 and 3). Daily parasitism decreased over the lifespan of T. remus females, except at 5 C and 6 days of storage (Fig. 3c). A total of 80% of S. frugiperda eggs (lifetime parasitism) were parasitized within 4 days (Fig. 2). However, adults stored at 10 C for 2 days achieved 80% of their lifetime parasitism on the fourth day, whereas adults stored at 10 C for 4 to 6 days reached 80% of their lifetime parasitism already on the second day (Fig. 2b d). Adults stored at 5 C for 2 and 6 days reached 80% of their lifetime parasitism on the fourth day (Fig. 3a and c), but on the second day when stored for 4 days (Fig. 3b). Discussion Overall, our results indicate that low temperature storage is possible for C. cephalonica eggs as well as T. remus pupae and adults. Storage can improve the parasitoid s availability to consumers, synchronize the desired developmental stage of the parasitoid to what is necessary for its release (decrease parasitoid development to reach the desired developmental stage at the desired day), increase efficiency, and provide standardized stocks for use in mass production (Leopold 1998). The higher tolerance to cold storage of viable C. cephalonica eggs compared with sterilized eggs may be due to an alteration of water content in eggs after exposure to ultraviolet light for sterilization, which can increase egg dehydration. However, eggs of Anagasta kuehniella (Zeller) (Lepidoptera: Pyralidae) irradiated under ultraviolet light for 45 min and stored between 1 and 4 C were equal or slightly

6 Queiroz et al Fig. 1 Daily and cumulative parasitism by Telenomus remus adults in eggs of Spodoptera frugiperda after different periods of Telenomus remus pupal storage. a 25 C for 0 days; b 5 C for 7 days; c 5 C for 14 days; d 10 C for 7 days; and e 10 C for 14 days. Arrows indicate 80% parasitism. superior in quality to freshly laid eggs to parasitism of Trichogramma evanescens (Westwood) or Trichogramma brasiliensis (Ashmead) (Hymenoptera: Trichogrammatidae) (Voegelé et al 1974). These differences may be due to differences in host egg structure: eggs of C. cephalonica have a rough texture, although not as rough as A. kuehniella eggs (Cônsoli et al 1999), and the egg membrane (chorion) of A. kuehniella eggs has a smaller number of pores and thin layers of mucous/wax to reduce dehydration (Cônsoli et al 1999), which might increase the tolerance to ultraviolet light and/or cold storage compared with C. cephalonica eggs. However, this hypothesis still needs confirmation and requires more studies to establish the relationship between egg sterilization techniques and low temperature storage. Interestingly, our results show that the parasitism rate of T. remus increased significantly for viable C. cephalonica eggs storage at 10 C and was higher than for fresh viable eggs, possibly because hardness and thickness of the egg chorion of C. cephalonica are modified during storage. Thus, the number of eggs that T. remus females was able to penetrate may be lower in fresh eggs, resulting in a low parasitism rate. In addition, stored viable C. cephalonica eggs do not readily lose water as they get older due to the chorion thickness and may therefore be more easily parasitized by T. remus. Similar results have been reported for Trissolcus semistriatus (Nees) (Hymenoptera: Platygastridae) parasitizing eggs of Eurydema ornatum (Linnaeus) (Hemiptera: Pentatomidae)

7 Low Temperature Storage of Telenomus remus and Corcyra cephalonica Table 4 Number of live adult male and female Telenomus remus produced after storage at different temperatures and for different times. Treatments Live males Live females Temperature of storage Days of storage 5 C 2 days 4.4 ± 0.2a 4.4 ± 0.2a 5 C 4 days 4.4 ± 0.2a 4.4 ± 0.2a 5 C 6 days 2.1 ± 0.3c 4.0 ± 0.2a 5 C 8 days 0.4 ± 0.2d 2.2 ± 0.5b 10 C 2 days 4.0 ± 0.3a 4.8 ± 0.1a 10 C 4 days 3.9 ± 0.4ab 3.9 ± 0.1a 10 C 6 days 2.6 ± 0.4bc 4.6 ± 0.4a 10 C 8 days 1.9 ± 0.3c 4.2 ± 0.1a CV (%) P < < F Means ± SEM followed by the same letter in a column do not differ statistically (Tukey test, P 0.05) (Kivan & Kilic 2005), indicating that this phenomenon occurs in different parasitoid species. The decrease in the number of C. cephalonica eggs parasitized by T. remus after a storage period of more than 14 days may be related to a loss of organismal properties favorable for parasitoid development, such as dehydration of eggs. This was reported for stored eggs of A. kuehniella offered to Trichogramma minutum (Riley) (Hymenoptera: Trichogrammatidae) (Van Steenburg 1934). After parasitizing stored C. cephalonica eggs, adult emergence of the T. remus offspring varied significantly across the different temperatures and storage periods. Many factors can interfere with parasitoid emergence (Foerster & Doetzer 2006). Long-term storage has negative effects on egg nutrients and can also change the water content of the host egg. However, our findings indicate that in most cases egg storage does not affect the emergence of T. remus adults when compared with the control. Storage of C. cephalonica eggs did not affect the sex ratio of T. remus offspring. Higher numbers of females than males are considered important in biological control programs because males do not contribute to parasitism-induced declines in pest populations (Navarro 1998). Therefore, our results suggest that storage of C. cephalonica eggs did not negatively impact biological control, since more females than males were still obtained from all treatments. It is also noteworthy that the length of the storage period of C. cephalonica eggs did not affect the sex ratio of T. remus, with a minimum value of This ratio was considered to be of acceptable quality in mass production of the parasitoid Trichogramma pratissolii (Querino & Zucchi) (Hymenoptera: Trichogrammatidae) (van Lenteren 2003). A major difficulty for the mass rearing of parasitoids in the laboratory, in addition to host availability, is their release immediately after adult emergence, due to the adults short life span. Therefore, apart from the storage of host eggs, the development of strategies for parasitoid storage (pupae or adult) is crucial in order to reduce parasitoid production costs and to improve its availability to consumers (Leopold 1998, Gardner et al 2012). It was believed that parasitoid pupae Table 5 Parasitism capacity of Telenomus remus females after storage at different temperatures and for different storage periods. Treatments Temperature of storage Days of storage Number of parasitized eggs Egg-to-adult period (days) Parasitoid emergence (%) Parental female longevity (days) Sex ratio none 87.5 ± 15.3bc 12.9 ± 0.0 ns 98.9 ± 1.0 ns 7.7 ± 1.4 ns 0.7 ± 0.1 ns 5 C 2 days ± 23.3a 13.0 ± ± ± ± C 4 days 40.0 ± 7.0c 13.0 ± ± ± ± C 6 days 42.5 ± 6.6c 12.3 ± ± ± ± C 8 days 30.0 ± 6.5c 13.0 ± ± ± ± C 2 days 58.5 ± 3.0c 13.0 ± ± ± ± C 4 days ± 15.0b 13.0 ± ± ± ± C 6 days 61.3 ± 0.8bc 12.5 ± ± ± ± C 8 days 36.8 ± 6.3c 13.0 ± ± ± ± 0.0 CV (%) P < F Means ± SEM followed by the same letter in a column do not differ statistically (Tukey test, P 0.05). Control treatments (no stored adult). ns ANOVA not significant

8 Queiroz et al Fig. 2 Daily and cumulative parasitism by Telenomus remus adults in eggs of Spodoptera frugiperda after different periods of Telenomus remus adult storage. a 25 C for 0 days; b 10 C for 2 days; c 10 C for 4 days; d 10 C for 6 days; and e 10 C for 8 days. Arrows indicate 80% parasitism. could be stored before adult emergence (Carvalho et al 2008), but it is important to assess the quality of the insects produced as well as the damage that such storage may cause to stored adults. Similar to host storage, the storage of T. remus adults had no direct impact on the sex ratio of their offspring. Similar findings for Telenomus busseolae (Gahan) (Hymenoptera: Platygastridae) are reported in the literature (Bayram et al 2005), indicating that long-term storage of parasitoid adults is possible. However, for T. remus pupae, we observed a downward trend of the secondary sex ratio with increasing storage time at 5 and 10 C, indicating that long-term storage of T. remus pupae impacts the sex ratio of the emerging parasitoids. This is of great concern for storage practices, since the number of females must be maximized for parasitoid release (Hassan et al 1990, Tezze & Botto 2004). Over the first 7 days of pupal storage, however, the sex ratio of the emerging offspring was not affected. Our analysis of the average survival of adult males and females of T. remus after storage at temperatures of 5 and 10 C showed that males have less tolerance toward storage than females. This may be due to males emerging earlier than females (Cave 2000) and having an overall shorter life span. In addition, a higher storage tolerance of females could be related to their larger body size, which may contribute to their longer survival at low temperatures of 5 and 10 C. Similar observations have been made while evaluating the storage of the parasitoid Gonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae) for 10 days at temperatures of 2, 5, and 10 C, noting that females survive longer than males

9 Low Temperature Storage of Telenomus remus and Corcyra cephalonica Fig. 3 Daily and cumulative parasitism by Telenomus remus adults in eggs of Spodoptera frugiperda after different periods of T. remus adult storage at 5 C. a 5 C for 2 days; b 5 C for 4 days; c 5 C for 6 days; and d 5 C for 8 days. Arrows indicate 80% parasitism. (Chen & Leopold 2007). It is suggested that the depletion of energy reserves may cause death or low survival rates during prolonged storage periods (Storey & Storey 1988). It is important to note that in addition to affecting secondary sex ratio, storage at 5 and 10 C also decreased T. remus pupae survival rates. Survival was the greatest (86%) during 7 days of storage at 10 C. Similar results were obtained with pupal storage of Trissolcus basalis (Wollaston) and Telenomus podisi Ashmead (Ashmead) (Hymenoptera: Platygastridae) at 15 Cforupto25dayswithmorethan85%survivalforboth species (Nakama & Foerster 2001). Similar to pupal survival, our results confirm that pupae of T. remus can be stored for 7 days without reducing the parasitism capacity of adults emerging from these pupae. However, 14 days of storage at 5 and 10 C impaired parasitism capacity. The reduction in the number of parasitized eggs may be related to the lower fertility of T. remus females resulting from an increased pupae storage time, a phenomenon previously reported for pupae of the parasitoid T. evanescens that had been stored for more than 3 weeks at 4 C (Ayvaz et al 2008). Similar results were also reported for pupae of Trichogramma oleae (Voegelé & Pointel) (Hymenoptera: Trichogrammatidae) stored for 3 weeks at 4 C (Gharbi 2014). Considering that survival is an important indicator of the effectiveness of insect storage (Bernardo et al 2008, Colinet & Boivin 2011), it is important to note that in our study, storage of T. remus pupae up to 7 days and storage of parasitoid adults did not affect survival, regardless of temperature. Storage of T. remus pupae for more than 7 days also impacted the parental longevity of emerged female adults, corroborating findings for Trichogramma nerudai (Pintureau & Gerding) (Hymenoptera: Trichogrammatidae) (Tezze & Botto 2004). Their study as well as that of Bernardo et al (2008) found that storage temperatures well below optimum produced adults with less body reserves and consequently reduced life span. Reduced longevity indicates susceptibility to cold storage of pupae over time. In contrast, storage of adult females did not impact their longevity. However, we did not observe such changes over time for T. remus females stored at 5 and 10 C. Storage of T. remus adults and pupae also did not impact parasitoid emergence of their offspring. Parasitoid emergence of the progeny of adults that had been stored as pupae (above 88%) or as adults (above 90%) was high (Bueno

10 Queiroz et al et al 2008). According to Pereira et al (2009), adult emergence after storage is an essential parameter for the successful use of the parasitoid. In this respect, we found that parasitoid emergence did not decline with increasing storage time, suggesting that it was not affected by the tested storage periods and temperatures. With respect to parasitism capacity by adults reared from pupae stored for different time periods, we found that parasitism was higher during the first 24 h, but decreased with increasing storage time. Possibly, there is a reduction of body fat reserves during storage at low temperatures affecting the performance of adults with respect to body mobility and consequently affecting parasitism (Couillien & Grégoire 1994). This could explain the decrease in T. remus parasitism capacity in the first 24 h. Using layered egg masses of S. frugiperda, weobserved parasitism in all layers suggesting that storage of T. remus pupae did not damage the parasitoid s ability to parasitize host eggs in overlapping layers. We obtained similar results when studying parasitism by stored adults over time. T. remus parasitism on eggs of S. frugiperda was highest in the first 24 h at both tested temperatures and for all periods of adult storage, also reported by Pomari et al (2012). Additionally, the similarity of total parasitism at both experimental temperatures (5 and 10 C) indicates that parasitoids stored at these temperatures suffered a reduction in their daily parasitism capacity, but not in total parasitism. Therefore, parasitoid storage can be considered a useful technique in mass parasitoid production. Overall, our results provide evidence that the damage on host eggs and parasitoids depends on temperature and storage duration. While C. cephalonica eggs can be stored at 10 C for until 21 days as viable eggs, the optimal storage period of the parasitoid (pupae and adults) is shorter. T. remus pupae can be stored at 10 C for until 7 days, and adults should not be stored for more than 4 days at either 5 or 10 C. The obtained knowledge on the limits of storage time for host and parasitoid will facilitate the development of a protocol and storage recommendations for this parasitoid rearing, improving its availability to consumers at lower prices. Acknowledgments The authors would like to thank Embrapa Soja, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number /2015-2, for funds that supported this research. Thanks are also extended to Dagmar Frisch for the English revision of this manuscript. This paper was approved for publication by the Editorial Board of Embrapa Soja. 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