Reproduction of Ooencyrtus submetallicus (Hymenoptera: Encyrtidae) and Trissolcus sp. aff. urichi (Hymenoptera: Scelionidae) in Eggs of Nezara viridula (Hemiptera: Pentatomidae) of Different Ages

The study of the interaction between parasitoid and host, especially the age of these organisms, is an important step towards the implementation of biological control programs. Therefore, we investigated the performance of Ooencyrtus submetallicus (Hymenoptera: Encyrtidae) and Trissolcus sp. aff. urichi (Hymenoptera: Scelionidae) parasitizing eggs of Nezara viridula (Hemiptera: Pentatomidae), considering different ages of the parasitoids and the host. We performed four laboratory bioassays: two using females of O. submetallicus and Trissolcus sp. aff. urichi at 24, 48, 72, 96, 120, or 144 hours of age exposed to parasitism in N. viridula eggs (24 h) and two trials with N. viridula eggs at 24, 48, 72, 96, 120, or 144 hours exposed to the parasitism of O. submetallicus and Trissolcus sp. aff. urichi (24 h). We evaluated the percentage of parasitism and emergence, life cycle length, progeny, sex ratio, and the longevity of the parasitoids. The parasitism of O. submetallicus in N. viridula eggs was influenced by the age of the parasitoid, 120 hours being the minimum to obtain better parasitism. From this age on, there is interference in the longevity of the progeny. Trisolcus sp. aff. urichi, at all ages, parasitized N. viridula eggs relatively well, but with almost no emergence of the parasitized eggs. Females of O. submetallicus parasitized and developed in eggs of N. viridula of all ages. Females of Trissolcus sp. aff. urichi parasitized their host, but there was barely any emergence. These pieces of information regarding the breeding methodology contribute to the implementation of new protocols for the multiplication of these parasitoids in the laboratory, and later, their release in the field.


Introduction
Biological control is an environmentally friendly and affordable management alternative, adopted to reduce insect populations considered pests for their natural enemies (Parra et al., 2002). In addition, this type of control is used to minimize excessive applications of synthetic insecticides, being a viable tool when associated with other strategies adopted by Integrated Pest Management (IPM) (Parra, 2014;Van Lenteren et al., 2018).
For the success of a biological control program, studies involving the interaction between the natural enemy and its host are needed, in order to understand the biology of these organisms and then adopt them as biological control agents (Parra & Zucchi, 2004;Siqueira et al., 2012). Several egg parasitoids have been reported, studied, and used as biological control agents (Crouzel & Saini, 1981;Corrêa-Ferreira & Moscardi, 1996;Queiroz et al., 2018;Martel et al., 2019;Stahl et al., 2019;Zerbino & Panizzi, 2019;Scaccini et al., 2020).
The green stink bug and other species of the complex group of phytophagous of the Pentatomidae family cause irreversible damage to the soybean crops from the beginning of pod formation to the final grain filling stage, affecting both yield and final quality (Panizzi & Slansky Jr, 1985;Soares et al., 2018). Commonly, species of the Pentatomidae complex are controlled with synthetic insecticides, both in seed treatment and in aerial application at the initial stages of crop development (Chiesa et al., 2016). However, this management method has been associated with the emergence of insect populations resistant to the main groups of insecticides adopted, the reduction of natural control agents, as well as other implications for the agroecosystem (Sosa-Gómez et al., 2001;Sosa-Gómez et al., 2010, Turchen et al., 2016. Therefore, having knowledge and development of research involving the interaction between the parasitoid and the host is necessary to increase the efficiency and success of biological control programs (Parra & Zucchi, 2004;Parra et al., 2019). Among these interactions, studying the age of both parasitoid and host females is not only important for analyzing the performance of biological control agents but also for predicting their establishment in the field (Cingolani et al., 2014;Queiroz et al., 2019;Hill et al., 2019;Queiroz et al., 2020a). Considering this, we determined the age of the parasitoids O. submetallicus and Trissolcus sp. aff. urichi that are considered optimal for parasitism in eggs of N. viridula. Furthermore, we evaluated the influence of the host egg age on the multiplication of both parasitoids and the influence of these factors on their biological characteristics. Breeding of O. submetallicus, Trissolcus sp. aff. urichi and N. viridula The experiments were set up based on stock cultures of the parasitoids O. submetallicus and Trissolcus sp. aff. urichi and host N. viridula, kept in an acclimatized room with temperature of 25±2 ºC, relative humidity of 70±10% RH, and a 12-hour photophase. The parasitoids O. submetallicus and Trissolcus sp. aff. urichi were identified according to Noyes (2010) and Johnson (1987), respectively. Voucher specimens were deposited at Coleção de Insetos Entomófagos "Oscar Monte" (Instituto Biológico, Campinas, SP, Brazil; curator Valmir A. Costa).

Laboratory
Nymphs and adults of N. viridula were placed in a 5 L transparent plastic containers and fed with fresh pods of Phaseolus vulgaris (Fabaceae), seeds of Ligustrum sp. (Oleaceae), raw grains of Arachis hypogaea L. (Fabaceae), and dry grains of Glycine max (Fabaceae) (Silva et al., 2008). In addition to portions of cotton moistened in distilled water to supply the insect's water needs. Filter paper (30 cm × 30 cm) folded in a fan shape and voile fabric (30 cm × 30 cm) were placed as a substrate for the stink bugs' oviposition. Adults of O. submetallicus and Trissolcus sp. aff. urichi were kept in glass tubes (15 cm high × 2 cm in diameter) closed with plastic film, fed with a droplet of honey (100%), and then multiplied into egg masses of N. viridula and Chinavia pengue (Rolston) (Hemiptera: Pentatomidae), respectively. The identification of the species N. viridula and C. pengue was carried out by Dr. Jocélia Grazia, (Universidade Federal do Rio Grande do Sul) Porto Alegre, Rio Grande do Sul. aff. urichi, and they were kept separately. These eggs were placed on cardboard cartons (1 × 5 cm), fixed with 20% gum arabic, and inserted into glass tubes (2 cm in diameter × 15 cm in height) ( Figure 1A). After 24 hours of parasitism, the females of the parasitoids were removed from the tubes and the hosts were transferred to the climatized chamber (BOD) at 25±2 ºC, 70±10% relative humidity, and 12-hour photophase until the emergence of the parasitoid adults. Five eggs of the host N. viridula (yellow and not deformed) at 24, 48, 72, 96, 120, or 144 hours of age were placed on cardboard sheets (1 × 5 cm), fixed with 20% gum arabic, and inserted in glass tubes (2 cm in diameter × 15 cm in height). Later, they were offered to females of O. submetallicus and Trissolcus sp. aff. urichi, both at 24 hours of age, in separate tubes ( Figure 1B). After 24 hours of parasitism, the females of each species were removed from the tubes and the cardboards containing the host's eggs were transferred to a climatized chamber (BOD) at 25±2 ºC, 70±10% relative humidity, and a 12-hour photophase until possible emergence of adults of the parasitoids.   Note. n.s. = not significant (p > 0.05); ¹ total egg duration-adult; ² number of individuals per egg; ³ n° of females/(n° of males + n° of females).

Discussion
In general, it was observed that the percentage of O. submetallicus parasitism increased according to the age of the females. This fact is easily explained because the representatives of the Encytidae family present synovigenic maturation, that is, the eggs are produced and, consequently, matured throughout their adult life and in general the females need a few days to start oviposition (Flanders, 1950;Papaj, 2000;Kapranas & Tena, 2015). Similar to our study, Aung et al. (2012) and Tunca (2016) verified an increase in the reproductive efficiency of Encyrtidae parasitoids from their fifth day of life.
Unlike O. submetallicus, the female ages of the parasitoid Trissolcus sp. aff. urichi did not influence the percentage of parasitism, remaining constant during the six days, possibly due to the viability of spermatozoa, guaranteed by the female's spermatheca during her reproductive phase (Damiens et al., 2003;Pascini & Martins, 2017). Regarding the age of the eggs of the host N. viridula, this factor was not significant for the percentage of parasitism, once females of O. submetallicus parasitized eggs up to 144 hours of age, as verified by Binazzi et al. (2013), where the parasitoid Ooencyrtus pityocampae (Mercet) (Hymenoptera Encyrtidae) parasitized eggs at up to seven days old (168 h), with no interference in their reproductive capacity. The host age did not generate any significant response in the parasitism of Trissolcus sp. aff. urichi, remaining above 80% at all ages of N. viridula eggs, however, some Scelionidae parasitoids prefer younger eggs for reproduction, because as the eggs age their embryonic content may change, compromising the quality of their nutritional status (Peñaflor et al., 2012;Da Wedge et al., 2017).
The percentage of emergence was not influenced by the ages of O. submetallicus, presenting a rate above 85%, as observed by Lee (1979) in his study on O. submetallicus and N. viridula. Similar to the age of the parasitoid, the various ages of N. viridula eggs did not cause variations in emergence since O. submetallicus emerged in more than 90% of the eggs at all ages of the host, similarly to what was described by Catalán and Verdú (2005), through the emergence of the progeny of the parasitoid Encyrtidae in more than 80% of the parasitized eggs of N. viridula.
Despite the multiple ages of Trissolcus sp. aff. urichi and N. viridula eggs not interfering in the percentage of emergence, few adults emerged in these bioassays. Possibly, the host does not have characteristics considered nutritionally suitable for the perfect development of the progenies, or even, due to non-reproductive effects such as the abortion of the parasitoid's eggs performed by the host's own immune system (Strand & Pech, 1995;Roversi, 2017;Abram et al., 2019). Botch and Delfosse (2018) also verified low percentage of the emergence of the parasitoid Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae) in eggs of non-target Pentatomidae hosts, and the authors associated this factor to the absence of interaction and/or non-suitability of the parasitoid to the host.
Both the age of O. submetallicus females and the age of N. viridula eggs did not interfere in the life cycle length of this parasitoid or in the number of individuals per egg, discriminating that the parasitoid can develop normally regardless of the age of the progenitor or host, which is similar to what was observed by Power et al. (2020) who, when studying alternative hosts for the reproduction of Ooencyrtus mirus (Triapitsyn and Power) (Hymenoptera: Encyrtidae), noted that the development time of this parasitoid in N. viridula eggs was approximately 15 days, being a suitable host for the reproduction of this parasitoid Encyrtidae. In our study, there was the emergence of more than one individual per egg, possibly due to the low density of eggs provided by females of O. submetallicus, with more than one adult emerging from a single host (Böckmann et al., 2012;Tunca et al., 2017). However, superparasitism tends to to lead to intraspecific competition within the host, which limits the nutrition of the progeny and can compromise the development of these parasitoids (Tunca et al., 2017).
The ages of Trissolcus sp. aff. urichi and N. viridula eggs did not influence the life cycle, the number of individuals per egg, and the longevity of males and females of this parasitoid, however, these characteristics were compromised by the non-target host, possibly due to its limited nutritional resources, inadequate egg size or even by the barriers of its immune system, as already mentioned (Vinson, 1976;Strand & Vinson, 1983;Abram et al., 2019). Other authors also report that T. urichi does not always develop in N. viridula eggs (Sujii et al., 2002), although its biological characteristics may undergo variations when subjected to other hosts of the Pentatomidae family (Laumann et al., , 2010Barakat et al., 2020).
The longevity of O. submetallicus progeny decreased as the age of the female increased, because as the parasitoid females age, there is a limitation of their nutritional resources that would be destined to the process of ovogenesis, resulting in the deficiency of proteins, carbohydrates, and lipids in their eggs (Giron & Casas, 2003;Muller et al., 2017). Therefore, the reduction in the number of eggs resulting from the increase in the age of the female progenitor may affect the process of nutrition of the offspring, thereby decreasing their aptitude and longevity (Lansing, 1947;Kindsvater & Otto, 2014;Plaistow et al., 2015;Muller et al., 2017). Unlike this, the age of the eggs of N. viridula did not have any effect on the longevity of O. submetallicus and it survived more than ten days, however, when compared to other host species and submitted to different diets, the longevity of the parasitoid may suffer several variations (Roversi et al., 2018).
Regardless of the age of O. submetallicus or N. viridula eggs, the sex ratio was characterized by the absolute presence of female individuals. The parasitoid O. submetallicus reproduces by thelytokous parthenogenesis, generating only female parasitoids at temperatures below 29.44 °C, however, males can also be produced at particular temperature variations (Wilson & Wooloo, 1952). This characteristic was also not influenced by the ages of Trissolcus sp. aff. urichi and neither by the different ages of the eggs of N. viridula, being characterized by the presence of male and female individuals, which is similar to what was observed by Queiroz et al. (2020b) through the emergence of individuals of both sexes of T. urichi in eggs of Pentatomidae hosts. From the results obtained and the discussion raised, this work provides grounds and opens the possibility for future studies about other stink bugs of the Pentatomidae complex, with the possibility of implementing new protocols for multiplication and for subsequent release of these species of parasitoids in a synchronized manner with the occurrence of stink bug populations in the field.