Biological Characteristics and Parasitism Capacity of Telenomus podisi ( Hymenoptera : Platygastridae ) on Eggs of Euschistus heros ( Hemiptera : Pentatomidae )

We evaluated the effects of adult mating and temperature on the biological potential and parasitism of Telenomus podisi (Ashmead) (Hymenoptera: Platygastridae) on Euschistus heros (Fabricius) (Hemiptera: Pentatomidae). Females were isolated and the characteristics of the F1 generation were compared with those of females previously copulated. The parasitism capacity of T. podisi on E. heros eggs at 20, 25, 30, 35, and 40 °C was evaluated. The longevity of parental females (days), egg-adult period (days) of the F1 generation, parasitism of parental females (%), and parasitoid emergence (%) were not influenced by the lack or not of the copulation. The offspring of non-copulated females were males, and arrhenotokous parthenogenesis occurred. The temperatures most favorable to the parasitoid were 25 and 30 °C. Even previously copulated females generated mostly males after a few days of oviposition. Therefore, the biological characteristics and reproduction of T. podisi were impacted by adult mating prior to parasitism and by different temperatures. Although parasitism of T. podisi was not affected by mating parasitoid permanence in the field, it may be affected when unmated females are released.


Introduction
Piercing-sucking insects are one of the most important groups of arthropods that attack soybeans in South America.Those pests are noteworthy for feeding directly on the pods, thereby seriously affecting crop yields (Panizzi & Slanky, 1985;Corrêa-Ferreira & Azevedo, 2002).In this complex of insects belonging to the family Pentatomidae, at least 54 species have been reported from soybean-growing areas (Panizzi & Slanky, 1985).Among them, the Neotropical brown stink bug, Euschistus heros (Fabricius) (Hemiptera: Pentatomidae), is the most abundant species, mainly in the central region of Brazil at latitudes between 0 and 23° (Panizzi & Corrêa-Ferreira, 1997).Thus, in order to mitigate losses caused by this pest and consequently increase profit, growers control this phytophagous arthropod (Zalucki et al., 2009).Currently, the primary method of pest control adopted by most soybean growers is the use of chemical insecticides (Song & Swinton, 2009;Panizzi, 2013).However, the abuse of insecticides has brought some negative effects (Song & Swinton, 2009), such as the rapid selection of pesticide-resistant pest strains that are resistant to a given active ingredient (Diez-Rodriguez & Omoto, 2001;Sosa-Gómez et al., 2001;Sosa-Gómez & Silva, 2010) or the reduction of natural biological control agents, especially when non-selective insecticides are used (Carmo et al., 2010;van Lenteren & Bueno, 2003).
In this scenario, the use of biological control as a more sustainable form of pest management is becoming more important (van Lenteren et al., 2017).Among the natural enemies used in biological control programs, the egg parasitoid Telenomus podisi (Ashmead) (Hymenoptera: Platygastridae) has shown great potential in parasitizing eggs of E. heros (Foerster & Queiroz, 1990;Corrêa-Ferreira & Azevedo, 2002;Doetzer & Foerster, 2007;van Lenteren et al., 2017).However, the success of T. podisi releases depends on an understanding of the bioecological characteristics of this parasitoid and on its interaction with the targeted host (Bourchier & Smith, 1996).Therefore, to validate the use of T. podisi in controlling E. heros in commercial releases, laboratory studies investigating parasitoid biology and its parasitism (Hassan, 1997;Scholler & Hassan, 2001) with biotic and abiotic factors are needed.Among those factors, mating and temperature might affect the biological characteristics of parasitoids, which could influence its efficiency in the field (Berti & Marcano, 1991;Mcdougall & Mills, 1997;Thomson & Hoffmann, 2002).
All biological parameters may be highly influenced by temperature (Noldus, 1989).Therefore, the study of egg parasitoid parasitism under different temperatures is of theoretical and practical interest, as it may provide crucial information for the establishment of biological control programs for E. heros in areas having similar temperatures as those tested.Each parasitoid species may present distinct behavior according to its adaptation to these temperatures, making it more or less suitable for a specific climate condition (Bleicher & Parra, 1989).Similarly, mating can also affect parasitoid performance (Sousa & Spence, 2000), since recently emerged adults will focus their energy first on mating and then on parasitism.Therefore, releasing mated or unmated females can differentially impact parasitoid efficacy in the field.Thus, the objective of the present study was to evaluate the effects of adult mating and temperature on the biological potential and parasitism of T. podisi on E. heros eggs.

Parasitoid and Host Colonies
Telenomus podisi females, as well as the host E. heros, used in this study originated from insect colonies kept at Embrapa Soybean, Londrina, State of Paraná, Brazil under controlled environmental conditions inside biochemical oxygen demand (BOD) climate chambers (ELETROLab®,model EL 212,São Paulo,SP,Brazil) set at 80±10% humidity, 25±2 °C, and a 14/10 h photoperiod (L/D).The insects were reared following proposed by Perez and Corrêa-Ferreira (2004) and Panizzi et al. (2000), and are briefly summarized as follows.
Telenomus podisi was originally collected in soybean fields in Londrina, State of Paraná.The population was maintained in the laboratory for approximately 5 years.In the laboratory, T. podisi was reared using eggs of E. heros glued to pieces of cardboard (2 × 8 cm) and introduced into tubes together with eggs already parasitized by T. podisi.Few drops of honey were placed inside these tubes to provide food for the adults as soon as they emerged.The tubes were then closed, and the eggs were allowed to be parasitized for 24 h.Adults emerging from these eggs were used for trials, as well as colony maintenance.
Euschistus heros was also originally collected from soybean plants in Londrina, State of Paraná.This population was maintained in the laboratory for approximately 2 years, during which new field insects were introduced each year to maintain insect quality.The insects were kept in cages (20 × 20 cm sides × 24 cm tall) made of a plastic screen and lined with filter paper.A Petri dish with a cotton wade soaked in distilled water (9 cm diameter) was added to each cage.Cages were cleaned, food was replaced, and egg masses were collected three times per week.After each collection, the egg masses were transferred to acrylic boxes (11 × 11 × 3.5 cm) lined with filter paper moistened with sterile-distilled water (Gerbox®).After eclosion, second-instar nymphs were transferred to new cages identical to those previously described.The laboratory-reared insects were then used for colony maintenance and to produce eggs for the trials.

Bioassay 1: Influence of Mating on Telenomus podisi Parasitism
The trial was carried out using a completely randomized experimental design with two treatments (unmated and mated females) and five replications, each composed of eight individual females (n = 40 females/treatment) inside glass tubes (12 × 75 mm tall).
To obtain unmated females, E. heros eggs were offered to the parasitoids for 24 h in 2 L plastic pots for parasitism, and then the eggs were placed in individual glass tubes (12 × 75 mm tall) until parasitoid emergence.After emergence, a droplet of honey was added to the glass tube to feed the adults.Some of the isolated females were placed with males for 24 h to copulate and some were left alone and remained unmated.
Each female was exposed to approximately 25 E. heros eggs, which were glued to white card (0.8 × 5 cm).The eggs were offered to the females for 24 h for parasitism.They were then removed and transferred to plastic bags (4 × 23 cm) where they remained until adult emergence.Both females and cards with parasitized eggs remained under the same conditions [temperature of 25±2 °C, 80±10% RH, and 14/10 h photoperiod (L/D)].The parameters evaluated included: the longevity of parental females (days), progeny egg-adult period (days), percentage of parasitism, emergence of parasitoids (%), and progeny sex ratio.

Bioassay 2: Telenomus podisi Parasitism Capacity on Eggs of Euschistus heros Under Different Temperatures
The trial was carried out in a completely randomized experimental design with five treatments (20, 25, 30, and 35±2 °C) and five replicates, each one composed of six individualized females (n = 30 females/treatment).Individual mated T. podisi (≤ 48-h old) were placed into separate glass tubes (12 ×75 mm tall) covered with PVC film.Droplets of pure honey on the walls of the glass tubes were offered to the females.Then, glass tubes (5 replications of 6 individualized females each) were prepared for each temperature.The tubes were then kept inside environmental chambers at temperatures of 20, 25, 30, and 35±2 °C, 8±10% RH, and 14/10 h photoperiod (L/D).Eggs (≤ 24-h old) from the host colony were frozen in nitrogen liquid and prior to the trial, were unfrozen and offered for parasitism for 24 h.These eggs were glued onto card (40 eggs each), on which the date and information of the treatment and replicate were recorded.These cards (with the eggs) were replaced daily until the death of the parasitoids.The eggs were removed daily from the glasses and maintained inside controlled environmental chambers under the same controlled conditions until the emergence of the parasitoids.The parameters recorded were: 1) the number of eggs parasitized per day; 2) the lifetime number of eggs parasitized per female; 3) parasitoid emergence (%); 4) progeny sex ratio; and 5) longevity of parental females (days).

Data Analysis
The results obtained in the experiments were submitted to exploratory analysis to evaluate the normality assumptions of the residuals (Shapiro & Wilk, 1965), homogeneity of variance of treatments, and additivity of the model to allow the application of ANOVA (Burr & Foster, 1972).The lifetime number of eggs parasitized per female and the longevity of adult parasitoids when presented with the host (in bioassay 2) were transformed by the emergence (%) of parasitized eggs by arcsine prior to the analysis of variance (ANOVA).Then, the averages were compared by Tukey test, at a 5% error probability, using the statistical analysis program SAS (SAS Institute, 2009).

Bioassay 1: Influence of Mating Status on Telenomus podisi Parasitism
Mating seemed to have a low effect on the evaluated characteristics of T. podisi parasitism.Longevity of parental females (days), progeny egg-adult period (days), parasitism (%), and emergence (%) did not differ between mated and unmated T. podisi females (Table 1).Only the progeny sex ratio differed between mated and unmated females, with 100% of progeny originating from virgin T. podisi females being males and only 46% of the progeny originating from the mated females being males, indicating the occurrence of arrhenotokous parthenogenesis in the absence of copulation (Table 1).Note. 1 Mean±standard error followed by the same letter in the columns did not differ by Tukey test (p > 0.05).ns ANOVA non-significant. jas.ccsenet.

Bioas Temperatu
The numb experimen parasitized number of 1).Higher of parasitis T. podisi r and 35 °C,  Based on the results observed in the present study, T. podisi present arrhenotokous parthenogenesis, since the absence of mating directly influenced the offspring sex ratio, leading to the production of haploid males only.
Reproduction via parthenogenesis of the arrhenotoky type has been described as the most common reproductive strategy for insects in the order Hymenoptera (Pratissoli et al., 2014).However, other biological parameters were not affected.The longevity of parental females, the progeny egg-adult period, parasitism capacity (%), and emergence of parasitoids from the parasitized eggs were similar between mated and unmated females.Queiroz et al. (2016) reported similar results for T. remus, where in progeny from mated females displayed a sex ratio of 0.6, while it was 0 for the unmated females.
Regarding the parasitism capacity of T. podisi, the results illustrate that temperature interferes in all evaluated biological aspects, as previously reported for other Platygastridae species (Yeargan, 1980;Nakama & Foerster, 2001).It is worth emphasizing that, although temperature is considered extremely important, other abiotic factors, such as relative humidity, may interfere with these biological characteristics (Parra, 1997;Pratissoli & Parra, 2001).Another point to be considered is the specific ability of T. podisi to introduce the ovipositor into the corium of the host eggs once these eggs lose their turgidity following a gradual increase in temperature (Bueno et al., 2009).The possible effects of temperature on this ability can affect the quantity and uniformity of T. podisi parasitism, which was not recorded in our study, and should be investigated in future.
Our results, show that the lifetime number of eggs parasitized per T. podisi female was lower at the highest studied temperature (35 °C).This value is directly related to the longevity of parental adult T. podisi females when presented with E. heros eggs.It was inversely correlated with temperature.The reduced longevity of parental adults owing to increased temperature has been previously reported for different parasitoid species (Maceda et al., 2003;Hansen & Jensen, 2002;Bourchier & Smith, 1996;Scholler & Hassan, 2001;Pratissoli & Parra, 2000;Pereira et al., 2004), and determines how long T. podisi is active in the field (how long T. podisi females are parasitizing host eggs).This might vary owing to differences in temperature (Reznik & Vaghina, 2006) or parasitoid species (Pratissoli & Parra, 2000;Pizzol et al., 2010).Thus, it might directly influence the success of biological control programs using egg parasitoids (Smith, 1996;Wajnberg & Hassan, 1994).
It is important to consider whether parasitism is more active during the first days of life or is evenly distributed throughout adulthood, when choosing the best parasitoid release strategy (Bueno et al., 2010).Then, those parasitoid characteristics should be considered when choosing the most suitable parasitoid species to be used in the field.The sooner the parasitoid reaches 80% of its lifetime parasitism, the better, because, while the parasitoids are exposed in the field, they might be susceptible to biotic and abiotic factors that can hinder their action.In practice, these factors might include fungicide or herbicide spraying that are necessary for crop management, or an abrupt change in temperature that can more easily kill the egg parasitoids (Carmo et al., 2010;Denis et al., 2011) but will rarely kill the pests.Egg parasitoids are usually small wasps, and are more susceptible to chemicals used in agriculture, such as herbicides and fungicides, than their hosts (Carmo et al., 2010).Furthermore, parasitoids differ from herbivorous insects by their usual inability to synthesize lipids as adults, which makes them more vulnerable to increases in temperature than most pests (Denis et al., 2011).
Although the number of parasitized eggs per day varied among the different temperatures, T. podisi oviposition peaked during the first 48 h of parasitism.Similar results were reported by Yeargan (1982) for T. podisi and Trissolcus euschisti (Ashmead) (Hymenoptera: Platygastridae) reared at 21 °C.This biological characteristic is not only observed in Trichogrammatidae, but also in Platygastridae.Peak oviposition of egg parasitoids from the genus Trichogramma has been reported during the first day after adult emergence for several different species of this genus (Pak & Oatman, 1982;Bai et al., 1992;Volkoff & Daumal, 1994).This is usually because most of these egg parasitoids have the capacity to store a full complement of mature eggs in the ovaries or oviducts, and complete oogenesis either before or shortly after adult emergence (pro-ovigenic parasitoids) (Mills & Kuhlmann, 2000) and, thus, adults emerge ready to lay eggs.In contrast, other studies have indicated that some parasitoid species emerge with an egg load that accounts for only a fraction of their potential parasitism, which exceeds their capacity to store mature eggs in their ovaries or oviducts (synovigenic parasitoids) (Houseweart et al., 1983;Smith & Hubbes, 1986;Kuhlmann & Mills, 1999).
Moreover, peak oviposition can vary as a consequence of pro-ovigenic or synovigenic relationships between oogenesis and oviposition, and also because of changes in parasitoid behavior (Denis et al., 2011).Adults of most Platygastridae species are incapable of lipogenesis; however, as an ectotherm insect, temperature is inversely related to their metabolic-rate and lipid consumption (Huey & Berrigan, 2001).Thus, in those species, the allocation of lipids accumulated during the larval stage determines adult lifespan and fecundity (Visser & Ellers, 2008) and, therefore, its lifetime reproductive success (Hyey & Berrigan, 2001).Because foraging decisions are affected by the state of external and internal variables, such as the lipid reserves and the number of mature eggs (Godfray, 1994) in females, lipids carried over from the larval stage can be allocated to either egg production or to adult lipid reserves, leading to a trade-off between reproduction and lifespan (Pexton & Mayhew, 2002).This confirms the importance of this knowledge with regards the developmental rate for widespread T. podisi augmentative release (Butler & Lopez, 1980).In this context, the higher number of parasitized eggs/day observed during the first 48 h in the present study is a positive characteristic of T. podisi.
The biological characteristics and reproduction of T. podisi were affected by adult mating prior to parasitism and different temperatures.Although parasitism of T. podisi, and therefore its control efficiency, was not affected by mating, parasitoid permanence in the field may be affected when unmated females are released.However, in augmentative biological control (ABC), parasitism capacity is more important than parasitoid maintenance in the field.Biocontrol agents are seasonally released in large numbers to rapidly reduce pests (van Lenteren et al., 2017); therefore, releasing mated females might not be important considering that the parasitism of available eggs in the field would not be affected (Carneiro et al., 2009).In addition, T. podisi exhibited high survival, as well as a high number of parasitized E. heros eggs, even at 30 °C.These results indicate that T. podisi might be well adapted to the studied temperature range (20 to 30 °C) and, thus, be potentially suitable for use in biological control programs of E. heros in different geographical areas within this range.However, it is important to emphasize that the results presented herein are from laboratory studies, and, therefore, additional field studies are needed to fully develop a biological control program using this egg parasitoid and to confirm the hypotheses presented here.

Conclusions
The biological characteristics and reproduction of T. podisi were influenced by adult mating prior to parasitism and by different temperatures.Although parasitism of T. podisi was not affected by mating parasitoid permanence in the field, it may be affected when unmated females are released.