ARTHROPOD BIOLOGY. Ann. Entomol. Soc. Am. 108(1): (2015); DOI: /aesa/sau002

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1 ARTHROPOD BIOLOGY The Importance of Relative Humidity for Telenomus remus (Hymenoptera: Platygastridae) Parasitism and Development on Corcyra cephalonica (Lepidoptera: Pyralidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Eggs ALINE POMARI-FERNANDES, 1,2 ANA PAULA DE QUEIROZ, 3 ADENEY DE FREITAS BUENO, 4 ALLISSON WILSON SANZOVO, 5 AND SERGIO ANTONIO DE BORTOLI 6 Ann. Entomol. Soc. Am. 108(1): (2015); DOI: /aesa/sau002 ABSTRACT The rearing of Telenomus remus (Nixon 1937) has been limited due to the difficulty of its development in its natural host, the eggs of Spodoptera frugiperda (J.E. Smith). Environmental conditions can strongly affect egg s parasitoid rearing on factitious hosts; however, until now only the effects of temperature have been studied. The importance of relative humidity in T. remus parasitism is poorly understood; therefore, this study evaluated its parasitism on Corcyra cephalonica (Stainton, 1866) and S. frugiperda eggs under different humidity regimes. Lifetime parasitism and parasitism viability were higher on S. frugiperda eggs and did not differ among humidity regimes. However, these biological parameters were higher at 80% humidity than at both 40 and 60% humidity when T. remus was reared on C. cephalonica eggs. The sex ratio was always higher than 0.6 for all hosts and humidity regimes. Parental female longevity was only influenced by humidity when presented to C. cephalonica eggs: it was higher at 60 and 80% humidity. T. remus egg-to-adult period was longer in C. cephalonica eggs; however, no difference between humidity regimes was noted. In general, relative humidity affected T. remus development only when it was reared on C. cephalonica eggs. For this factitious host, 80% humidity was shown to be appropriate for egg parasitoid rearing due to its better biological performance. KEY WORDS factitious host, biological control, egg parasitoid, parasitoid rearing Introduction Telenomus remus (Nixon 1937) (Hymenoptera: Platygastridae) is an egg parasitoid of various species of the order Lepidoptera, many of which are crop pests around the world. This parasitoid has a high reproductive capacity, and is an effective biological control agent against pests, particularly of those from the genus Spodoptera (Cave 2000, Pomari et al. 2012). Each parasitoid female produces 270 eggs during its lifespan, usually laid individually on each host egg; therefore, superparasitism is rare for this species (Cave 2000). T. remus is noteworthy for effectively parasitizing the eggs of Spodoptera spp., in superposed layers, even parasitizing eggs located in the inner layers of the egg mass (Figueiredo et al. 1999, Bueno et al. 2008), a property 1 Universidade Federal da Fronteira Sul, BR 158, Km 405, Caixa Postal 106, Laranjeiras do Sul, Paraná, Brasil, Corresponding author, aline.fernandes@uffs.edu.br. 3 Instituto Agronômico do Paraná, Rodovia Celso Garcia Cid, Km 375, Londrina, Paraná, Brasil, Empresa Brasileira de Pesquisa Agropecuária - Embrapa Soja, Caixa Postal 231, Londrina, Paraná, Brasil, Universidade Estadual do Norte do Paraná, Cornélio Procópio, Paraná, Brasil, Universidade Estadual Paulista, Faculdade de Ciências Agrárias de Jaboticabal, Jaboticabal, São Paulo, Brasil, rarely recorded for other egg parasitoid species. Additionally, T. remus has high dispersal and host search capacities (Pomari et al. 2013a,b), underlining its potential for augmentative biological control programs. Despite all of these favorable features for biological control, this parasitoid is currently only reared on a small scale due to the difficulties inherent in rearing it on its natural host, Spodoptera frugiperda (J.E. Smith). Therefore, this parasitoid has been only used for experimental purposes against S. frugiperda and other Lepidoptera. An effective and inexpensive mass-rearing method for T. remus in laboratories or bioindustries is a crucial step toward the success of biological control programs using this species. A possible solution to this problem might be the use of a factitious host. It is important to point out that there are biological control agents that are reared on natural hosts, often in a labor intensive and expensive way, which can be financially unviable for use in the field (Parra 1997). However, there are also natural enemies that can be reared on factitious hosts that often reduce production costs, increasing their viability for large-scale use (Parra 1997). T. remus development in the eggs of Corcyra cephalonica (Stainton, 1866) (Lepidoptera: Pyralidae), a stored-product moth, has been reported by Cave (2000), making this moth a factitious host candidate. However, the quality of the insects produced in the VC The Authors Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com

2 12 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 108, no. 1 laboratory is directly associated with the environmental conditions under which they are kept. Among these environmental conditions, temperature and humidity seem to be the most important (Cave 2000). Temperature effects on T. remus parasitism and development was extensively studied by Pomari et al. (2012) in the eggs of four species of the genus Spodoptera. In contrast, the importance of relative humidity (RH) in T. remus parasitism and development is poorly understood to date. Therefore, the aim of this research was to study the importance of relative humidity in the development of T. remus on both C. cephalonica and S. frugiperda eggs, evaluating parasitoid biology and parasitism capacity. Materials and Methods Parasitoid and host colonies. C. cephalonica and S. frugiperda eggs and T. remus femalesusedinthe experiments were from insect colonies kept at Embrapa Soybean, Londrina, State of Paraná, Brazil. S. frugiperda was originally collected from maize plants in Rio Verde, State of Goiás, and had been kept in the laboratory for approximately five years. They were reared under laboratory-controlled environmental conditions ( C temperature, % RH, and a photoperiod of 14:10 [L:D] h) and fed on an artificial diet described by Greene et al. (1976) and Parra (2001). C. cephalonica was collected from UNESP/Jaboticabal and had been kept in the laboratory for approximately three years. C. cephalonica was reared on its natural diet, using a methodology adapted from rearing Anagasta kuehniella (Lepidoptera: Pyralidae) (Parra 1997). T. remus was originally collected in Ecuador and grown at the parasitoid rearing facilities of ESALQ/ USP (Luiz de Queiroz College of Agriculture/University of São Paulo), from where some specimens were transferred to Embrapa Soybean seven years ago. In the laboratory, T. remus was reared using S. frugiperda egg masses (150 eggs each), which were glued onto a cardboard sheet (2 by 8 cm 2 ) and placed with eggs previously parasitized by T. remus. Small drops of honey were placed inside these tubes to feed the adults as soon as they emerged. The tubes were then closed, and the eggs allowed to be parasitized for 24 h. The adults that emerged from these eggs were used for trials or colony maintenance. Before starting the experiment, T. remus was reared for five generations in the eggs of both S. frugiperda and C. cephalonica, to eliminate possible preimaginal conditioning by the successive rearing of T. remus in S. frugiperda eggs. Biological Characteristics of T. remus in C. cephalonica and S. frugiperda Eggs at Different Relative Humidity Regimes. The experimental design was fully randomized, with treatments arranged in a 2 by 3 factorial scheme (two host species and three humidity regimes), with four replicates per treatment. Each replicate comprised five T. remus females (up to 24 h old) and 100 fresh host eggs in an individual vial (12 mm in diameter and 75 mm in height). The host species and humidity regimes studied were C. cephalonica and S. frugiperda at , , and %. Controlled environmental conditions were obtained in biochemical oxygen demand climate chambers (ELETROLab, model EL 212, São Paulo, SP, Brazil) set at the desired humidity regimes, with a temperature of C and a photoperiod of 12:12 (L:D) h. T. remus females (up to 24 h old) from eggs of both hosts were placed in individual 12-mm-diameter by 75-mm-tall glass vials, each containing a honey droplet. Theeggs(upto24hold)ofeachhostweregluedonto 0.8 by 5 cm 2 rectangular cards of white bristol board paper labeled with the respective treatments. These cards were placed in individual vials, each containing a single T. remus female, and sealed with PVC film that had small pin holes on it enabling humidity exchange with the environment but not allowing parasitoids to escape. The T. remus females were allowed to parasitize for 24 h, then the cards were removed from the vials and transferred to new ones, which were exposed to the given humidity until the emergence of the adults. For each humidity and host, the following biological parameters were recorded: the number of eggs parasitized in 24 h, the duration of the egg adult period, the percentage of emergence (viability ¼ number of parasitized eggs with emergence holes/total number of parasitized eggs 100), which was possible counting all eggs with the aid of a stereomicroscope, and the sex ratio (female/(female þ male)). To determine the duration of the egg adult period, daily observations from emergence to the adult stage were made. T. remus Parasitism Capacity in C. cephalonica and S. frugiperda Eggs Exposed to Different Relative Humidities. Similar to the biological characteristics trial, this experiment was conducted with a fully randomized design with treatments arranged in a 2 by 3 factorial scheme (two host species and three humidity regimes), with four replicates per treatment. Each replicate, consisting of five T. remus females from each host (up to 24 h old), was exposed to humidities of , , and %. The trial was conducted under controlled environmental conditions in biochemical oxygen demand climate chambers set at the desired humidity, with a temperature of Candaphotoperiod of 12:12 (L:D) h. Each day of the trial, 100 eggs (up to 24 h old) from each host were glued onto 0.8 by 5 cm 2 rectangular cards of white Bristol board paper labeled with the respective treatments. The eggs were then allowed to be parasitized for 24 h. Eggs were exchanged daily until the death of the female parasitoid. The cards with the parasitized eggs were kept under the same conditions as those in a climatic chamber until the parasitoids emerged. The evaluated parameters were the number of parasitized eggs per day (daily parasitism), lifetime parasitism, and parental female longevity. Data Analyses. Before ANOVA, the experimental results were submitted to exploratory analysis to test the normality of the residuals (Shapiro and Wilk 1965), the homogeneity of variance of the treatments (Burr and Foster 1972), and the additivity of the model. Means were compared using the Tukey test (5% error

3 January 2015 POMARI-FERNANDES ET AL.: RH INFLUENCE ABOVE T. remus PARASITISM 13 probability) through the SAS statistical analysis program (SAS Institute 2001). Results Biological Characteristics of T. remus in C. cephalonica and S. frugiperda Eggs at Different Relative Humidity Regimes. Values of parasitized eggs in 24 h by T. remus females (Table 1) and their parasitism viability (% emergence; Table 2) were significantly higher on S. frugiperda eggs than on C. cephalonica eggs under all humidity regimes (number of parasitized eggs: F 9, 20 ¼ 35.98, P < , Table 1; parasitism viability: F 9, 20 ¼ 14.50, P < , Table 2). However, relative humidity only influenced the results for C. cephalonica, with a higher number of parasitized eggs (Table 1) and emergence of the parasitoid (Table 2) when exposed to 80% humidity. The T. remus sex ratio only differed between the hosts at 80% humidity, with the highest number of females obtained from C. cephalonica (0.85). Less females (0.61) were obtained for S. frugiperda (Table 3). There was no interaction between humidity and the host in the factorial analyses for the period between Table 1. Number of C. cephalonica and S. frugiperda eggs parasitized by T. remus in 24 h, at different relative humidity, T: C, and a photoperiod of 12:12 (L:D) h C. cephalonica bAB bB bA S. frugiperda aA aA aA CV (%) Table 2. Parasitism viability of C. cephalonica and S. frugiperda eggs parasitized by T. remus, at different relative humidity, T: C and a photoperiod of 12:12 (L:D) h C. cephalonica bC bB bA S. frugiperda aA aA aA CV (%) 8.93 Table 3. Sex ratio of T. remus when reared on eggs of C. cephalonica and S. frugiperda parasitized at different relative humidity, T: C and a photoperiod of 12:12 (L:D) h C. cephalonica aA aA aA S. frugiperda aA aA bA CV (%) egg to adult. Therefore, there was no humidity effect on T. remus development. The mean egg-to-adult period was 12.5 d (Fig. 1A). This biological parameter was affected by the host, with a longer development period required on C. cephalonica eggs (Fig. 1B). T. remus Parasitism Capacity in C. cephalonica and S. frugiperda Eggs Exposed to Different Relative Humidity Regimes. Adult female longevity differed between the hosts only under 60 and 80% humidity (F 9, 20 ¼ 18.78, P < ). Females that parasitized the eggs of C. cephalonica lived longer, but at 40% humidity female longevity was lower when presented with C. cephalonica eggs (Table 4). Neither was there interaction between the factors humidity regime and host nor difference between humidity regimes regarding lifetime parasitism (Fig. 2A). However, lifetime parasitism did differ between hosts; the largest number of parasitized eggs was recorded in S. frugiperda (Fig. 2B). Daily parasitism decreased over the lifetime of the females (Fig. 3). For C. cephalonica, the greatest daily parasitism occurred in the first 24 h, when exposed to humidities of 40 and % (Fig. 3A and C). At 60% humidity, the greatest daily parasitism occurred in the first 120 h (Fig. 3B). The mean numbers of eggs parasitized were 6.04, 9.08, and 3.88 under , , and % humidities, respectively. Parasitism reached 80% at 6, 5, and 9 d under , , and % humidities, respectively (Fig. 3A C). For S. frugiperda, the greatest parasitism occurred in the first 24 h under all humidities (40, 60, and 80%), and the mean numbers of eggs parasitized were 64.65, 64.55, and 63.00, respectively. Parasitism reached 80% on the third day under all three relative humidities (Fig. 3D F). Discussion Temperature and relative humidity are the most important abiotic factors for parasitoid development, and consequently for their mass-rearing in laboratories or bioindustries (Cave 2000). The influence of temperature on T. remus parasitism has been studied, and an optimum temperature range (25 28 C) has been established for T. remus development (Bueno et al. 2008, Pomari et al. 2012). In contrast, the importance of relative humidity in T. remus parasitism is poorly understood, particularly when taking into consideration the factitious hosts such as C. cephalonica. To the best of our knowledge, this is the first report on T. remus parasitism on C. cephalonica eggs under different relative humidities that can offer the required knowledge to successfully rear T. remus on this factious host. ApositiveeffectofincreasedhumidityonT. remus parasitism on Spodoptera litura (F.) (Lepidoptera: Noctuidae) eggs has been previously reported. More than 90% parasitism was recorded when the relative humidity was between 50 75% (Gupta and Pawar 1985). Gautum (1986) found that the highest parasitism occurred at 75% RH on Agrotis spinifera (Hübner) (Lepidoptera: Noctuidae) eggs. Furthermore, higher parasitism and parasitism viability was recorded for

4 14 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 108, no. 1 Fig. 1. (A) Egg adult period (mean 6 SE) of T. remus at different relative humidity. (B) Egg adult period (mean 6 SE) of C. cephalonica and S. frugiperda eggs paratisized by T. remus. Treatments followed by the same letter are not statistically different by Tukey test (P 0.05). Table 4. Female longevity of T. remus that parasitized eggs of C. cephalonica and S. frugiperda at different relative humidity, T: C and a photoperiod of 12:12 (L:D) h C. cephalonica aB aA aA S. frugiperda aA bA bA CV (%) Telenomus isis (Polaszek) parasitizing coffee borer eggs, Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae), Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae), and Busseola fusca (Fuller) (Lepidoptera: Noctuidae) when the humidity was 70 80% compared with when it was 40 50% (Bruce et al. 2009). These results are similar to the parasitism recorded in this study for C. cephalonica eggs, but different for S. frugiperda eggs, as changes in humidity between 40% and 80 did not influence parasitism or the development of T. remus on its natural host (S. frugiperda). In contrast to what was observed for T. remus parasitizing S. frugiperda eggs, relative humidity had an influence on various biological parameters of the parasitoid in C. cephalonica eggs. This effect has not previously been documented; for example, Kumar et al. (1986) reported that a relative humidity of 70% was used to rear T. remus in this alternative host, without giving reasons for the choice of this value. Therefore, the effect of low humidity on the number of parasitized eggs, emergence percentage, and female longevity in the case of C. cephalonica indicates that T. remus requires higher humidity to optimize its development and parasitism when developed in this host egg. Egg chorion must provide protection against water loss, as well as have the necessary texture and hardness to be drilled, and cues that may elicit host recognition and acceptance by T. remus females. The shape, size, color, and chorionic structure of host eggs are key factors in the host selection process for various species of parasitoid (Salt 1938, Schmidt and Smith 1987, Pak et al. 1990, Schmidt 1994). C. cephalonica eggs have few aeropyles located near the joining ridges. Their chorion thickness ranges from 4.18 to 5.32 mm. In contrast, S. frugiperda eggs have a moderate number of aeropyles located at the intersection between bridges and ridges. Their chorion is 2.50 mm to mm thick, forming a discontinuous mucous layer (Cônsoli et al. 1999). The differences in the thickness of the chorion, particularly the exochorion (the most protein-rich layer), can affect not only the handling time and exploitation by parasitoids, but also host suitability to the parasitoid (Pak et al. 1990), as well as the water and oxygen balance with the environment (Hinton 1981). Sensitivity of host eggs to dehydration appears to increase with an increasing number of aeropyle openings, and decrease with an increasing thickness of the mucous layer (Cônsoli et al. 1999). Such relationships between the structure of the chorion and the sensitivity of the host egg to desiccation have been reported for many species of Lepidoptera (Barbier and Chauvin 1974, Chauvin and Chauvin 1980). However, differences in the number of aeropyle openings between C. cephalonica and S. frugiperda eggs may also be indicative of their metabolic rate. For egg parasitoids, there is only one study, to our knowledge, on the metabolic rate of the development of Trichogramma, which reported that oxygen consumption by Trichogramma dendrolimi Matsumara (Hymenoptera: Trichogramatidae) increased during the late larval and pupal stages, after the parasitoids had fed off the host (Dai et al. 1982). A large number of aeropyles in the eggs may indicate a high metabolic rate during embryonic development, and it may be related to oxygen availability for parasitoid development. The reduced host surface area for gas exchange, such as the case of C. cephalonica eggs compared with S. frugiperda eggs, may affect the development of parasitoid larvae. Early-stage Trichogramma, another egg parasitoid, can use dissolved oxygen available in the host egg, or oxygen stored in the trabecular layer (Cônsoli et al. 1999). Trichogramma at this stage does not have any specialized structure to make an opening in the chorion of the host to gain contact with atmospheric air; such specialized structure is found in the larva of the encyrtid mealy bug parasitoid (Nenon and Biassangama 1985). However, the low number of openings for gas exchange could impair the

5 January 2015 POMARI-FERNANDES ET AL.: RH INFLUENCE ABOVE T. remus PARASITISM 15 Fig. 2. (A) Number of parasitized eggs (mean 6 SE) by T. remus at different relative humidity. (B) Number of parasitized eggs (mean 6 SE) of C. cephalonica and S. frugiperda by T. remus. Treatments followed by the same letter are not statistically different by Tukey test (P 0.05). Fig. 3. Daily (number) and lifetime parasitism of C. cephalonica (A) UR: %, (B) UR: %, (C) UR: %; and S. frugiperda (D) UR: %, (E) UR: %, (F) UR: % eggs by T. remus.

6 16 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 108, no. 1 development of Trichogramma after its larval stage, when there is a high oxygen demand. In addition, Trichogramma has to access oxygen through the chorion of the host, unlike Edovum puttleri Grissell (Hymenoptera: Eulophidae), another egg parasitoid, that uses its mandibles to perforate the host chorion before becoming a pupa, to gain oxygen directly from the air (Laudonia and Viggiani 1986, Colazza and Bin 1992). The oxygen demand of T. remus and the mechanism by which this parasitoid controls its gas exchange are unknown. A better understanding of the effects of increasing environmental humidity on oxygen availability to the parasitoid is required, as it may play an important role in host suitability to parasitoid development. The difference in the effect of humidity on parasitism in the two hosts may be because of the physiological differences between them; as the factitious host is able to develop in a low humidity environment (15%; Cox et al. 1981), the eggs have a low water content, and an increase in humidity in this environment might be required for the proper development of the parasitoid. However, features like host egg volume, chorion thickness, nutritional content, age, and the shape of the host egg masses can affect the ability of the host egg to maintain its humidity, which affects parasitism and the sex ratio (Hoffmann et al. 2001, Roriz et al. 2006, Rukmowati-Brotodjojo and Walter 2006). With respect to sex ratio, it is important to highlight that this is an important biological parameter when considering massrearing for use in biological control programs (Bueno et al. 2008). This is because a greater number of females is desirable, because they are responsible for the direct suppression of target pests (Bueno et al. 2008), and because if the number of males and females is similar to that found in nature, high quality insects will be used (van Lenteren 2003). The sex ratio results obtained for the two hosts were similar, and favorable for the rearing aiming at parasitoid releases in the field, because of the higher number of females. However, it is noteworthy that the offspring sex ratio of T. remus can also be affected by female age, as Spodoptera eggs parasitized by females within 2 3 d of life have a sex ratio that is typically 60 70% female, but may decrease to 22% female with female age (Schwartz and Gerling 1974). Another factor that affects the ratio of males to females is the amount of available host eggs, because T. remus females produce more males when the number of parasitoids is much greater than the number of hosts (van Welzen and Waage 1987). Development (egg adult period) is dependent on different biotic and abiotic factors (Pomari et al. 2012). The host nutritional availability determines the need for a longer or shorter period of development. Most T. remus egg adult development in this study were longer on the eggs of C. cephalonica than on the eggs of S. frugiperda, indicating that this alternative host s eggs are nutritionally inferior to the natural host, resulting in a prolongation of the developmental period. Some authors have reported that the intrinsic characteristics of the egg may be important, as nutritional quality may be the key factor in the acceptance and development of the host (Shipp et al. 1998, Pratissoli and Parra 2001). Parental female longevity is correlated with the energy expenditure required for parasitism (Almeida 2004). Therefore, the lower number of parasitized eggs of C. cephalonica, coupled with the fact that this kind of posture be offered in a single layer, may have led to a lower metabolic rate, consequently increasing female longevity. Some authors have reported that for Trichogramma pretiosum this variability is related to the genetic, phenological, and physiological characteristics of the host (Lewis and Martin 1990). For other species of egg parasitoids, such as Trichogramma atopovirillia Oatman and Platner, the higher energy expenditure of females is due to parasitism, firstly with the introduction of the ovipositor and oviposition, followed by a review of the host by contact with the antennas (Almeida 2004). Besides these factors, one should take into account that T. remus females have the maximum numberofeggsintheirovariesbetween2 3dofage(van Welzen and Waage 1987), and produce >76% of their offspring during the first 5 d of adulthood (Schwartz and Gerling 1974). Thus, as seen in our experiments, females reached 80% parasitized in the first 5 d, so the longevity of female parental eggs, although important, is not a determining factor between the hosts, as parasitoids from both hosts lived longer than 5 d. The total number of S. frugiperda eggs parasitized per T. remus female was similar to that obtained by Pomari et al. (2013a,b) in all tested humidities. Moreover, there was no difference in this parameter between the two hosts, the lowest being C. cephalonica. This result can be directly related to preimaginal conditioning, which, according to (Cobert 1985), can occur when a parasitoid reproduces over successive generations in a particular host. Acknowledgments We thank Fapesp (Number Process 2011/ ), Embrapa Soja, and CNPq for the financial supporting that made this research possible. References Cited Almeida, R. 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