Biological characteristics of Trissolcus urichi (Crawford) (Hymenoptera: Scelionidae) on Euschistus heros (Fabricius) and Dichelops melacanthus (Dallas) (Hemiptera: Pentatomidae) Eggs

Species of the genus Trissolcus are effective as egg parasitoids of Euschistus heros and can potentially be used in a multispecies pest management approach. However, in order to successfully use those biocontrol agents in the field, previous detailed knowledge about their life history are necessary. Therefore, we evaluate some biological characteristics of Trissolcus urichi on Euschistus heros and Dichelops melacanthus eggs. Three independent experiments were performed: (1) T. urichi host preference between E. heros and D. melacanthus eggs. (2) T. urichi eggs-adult period (days), number of parasitized eggs in 24 h, emergence rate (%) and sex ratio of the parasitoid in E. heros and D. melacanthus eggs. (3) Morphometric characteristics of T. urichi grown on E. heros and D. melacanthus eggs. Trissolcus urichi preferred to parasitize E. heros eggs, exhibiting a higher number of parasitized eggs, higher rate of emergence (%) and faster development, as well as producing progeny of larger size than the parasitoids emerged from eggs of D. melacanthus in relation to body length, wing length and width. Thus, it can be concluded that T. urichi had better performance on E. heros eggs, although the parasitoid had also acceptable parasitism capacity and development in D. melacanthus eggs.

Scientific RepoRtS | (2020) 10:12441 | https://doi.org/10.1038/s41598-020-69406-z www.nature.com/scientificreports/ Current stink bug management strategies in the field are primarily based on the application of pesticides 9 . However, insecticide overuse has triggered problems such as increased production costs, elimination of existing natural enemies, selection of insecticide-resistant pests, and contamination of the environment [10][11][12] . Therefore, a more sustainable pest management approach is urgently needed. Among the most sustainable pest management tools available, augmentative biological control (ABC) stands out due to its efficacy and worldwide acceptance, being used on more than 30 million ha globally 13 .
Despite this potential parasitism on stink bugs eggs, it is important to consider that in field conditions, it is likely that foraging T. urichi individuals would encounter the eggs of one host species before the eggs of another due to temporal or spatial differences in the hosts' ovipositional activities. Therefore, this work studied T. urichi parasitism on E. heros and D. melacanthus eggs as well as parasitoid parasitism preference among those hosts.  22,23 and briefly summarized in the followings.

Material and methods
Trissolcus urichi was collected originally from soybean fields in Embrapa Soybean Experimental Farm, Londrina, Stated of Paraná, Brazil (23° 11′ 11.7" S and 51° 10′ 46.1" W). The colony has been kept in the laboratory for approximately 3 yr. It has been reared on E. heros eggs (aged ≤ 24 h) glued to pieces of card (5 cm × 8 cm). When parasitoid was close to emergence (1 day before), new eggs (aged ≤ 24 h) were introduced into plastic cages (8.5 cm high and 7 cm in diameter) together with the eggs already parasitized by T. urichi close to parasitoid emergence. Small drops of Apis mellifera-produced honey were placed inside these tubes to provide food for the adults when they emerged. The tubes were then closed, and after adult emergence, the eggs allowed to be parasitized for 24 h. After 24 h, the eggs recently parasitized were removed to other cages starting a new parasitoid cycle. Adults that emerge from these eggs were used for trials as well as for colony maintenance.
Stink bug species were originally collected in soybean (E. heros) and maize (D. melacanthus) fields also in Embrapa Soybean Experimental Farm, Londrina, State of Paraná, Brazil (23° 11′ 11.7" S and 51° 10′ 46.1" W). Those populations were kept in the laboratory for approximately 4 yr during which new field insects were introduced each yr to maintain colony quality. Those insects were kept in plastic screen cages (20 cm × 20 cm sides × 24 cm tall) (Plasvale Ltda., Gaspar, State of Santa Catarina, Brazil) lined with filter paper and fed ad libitum with a mixture of beans (Phaseolus vulgaris L.; Fabaceae), soybeans (Glycine max L. Merr.; Fabaceae), peanuts (Arachis hypogaea L.; Fabaceae), sunflower seeds (Helianthus annuus L.; Asteraceae) and privet fruits (Ligustrum lucidum Aiton; Oleaceae). A Petri dish (diameter 9 cm) with a cotton wad soaked in distilled water was added to each cage. Cages were cleaned, food replaced, and egg masses collected on a daily basis. The eggs were then used for trials or colony maintenance.
Trissolcus urichi host preference between eggs of E. heros and D. melacanthus (bioassay 1). The host preference test was performed in a completely randomized design, with two treatments (E. heros and D. melacanthus eggs) and 15 replicates, each one using one arena with a double chance of choice (Fig. 1). The arenas were adapted from those previously described in literature 24 , composed of polyethylene bottles (4 cm high and 2 cm in diameter) and two plastic microtubes (12 mm diameter × 75 mm height) arranged equidistant at the bottom of the bottle and a microtube (12 mm diameter × 75 mm height) arranged at the top of the arena ( Fig. 1) 25 .
Euschistus heros and D. melacanthus eggs; which have the average size of 0.83 mm width × 0.91 mm length for E. heros eggs and 0.82 mm width × 0.98 mm length for D. melacanthus eggs 25 ; were counted (40 eggs from each host) and placed on cards (1 cm × 6 cm), and introduced into each tube on opposite sides of the arena (Fig. 1). parasitism of E. heros and D. melacanthus eggs by T. urichi (bioassay 2). The parasitism experiment was conducted in a completely randomized design with two treatments (E. heros and D. melacanthus eggs) and four replicates (each replicate composed of five females). Newly emerged T. urichi females (≤ 48 h old, mated with no previous parasitism experience) were individually placed in microtubes (8 cm × 2 cm) and fed with a Apis mellifera-produced honey droplet. Forty host eggs were glued with white glue (Tenaz®) in white card (1 cm × 6 cm) identified according to the treatments. The cards were placed in the microtubes together with the T. urichi females and sealed with PVC film for a period of 24 h. After this period the females were removed and the eggs kept in the same BOD chamber for later evaluation. The biological parameters evaluated were the number of parasitized eggs, egg-adult development period (days), percentage of emergence, and sex ratio. Daily observations of progeny emergence were performed to determine the egg-adult period.

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Trissolcus urichi adult morphometry when reared on E. heros and D. melacanthus eggs (bioassay 3). The experiment was conducted in a completely randomized design in a 2 × 2 factorial scheme: parasitoids from two hosts (E. heros and D. melacanthus) × two genera of the parasitoid (male and female) and 10 replicates. Ten females and 10 males of T. urichi progeny were analyzed for each host. For each parasitoid, morphometric measurements of the right anterior wing length and width, right posterior tibia length, and body length (head to the end of the abdomen) were performed, according the standardized quality control procedures established by the International Organization of Biological Control (Global IOBC Working Group: 'Quality Control of Mass Reared Arthropods') 26 . For the evaluation of these morphological characters, each specimen was photographed with a stereoscopic microscope (Leica Application Suite -Version 1.6.0) and the morphometry measured using Image J (Version 1.47) 25 .

Data analysis
The results obtained in the experiments were submitted to exploratory analysis to evaluate the normality assumptions of the residues 27 , homogeneity of variance of treatments, and additivity of the model to allow the application of ANOVA 28 . The means were compared using the Tukey test, at 5% of error probability, using the statistical analysis program SAS 29 . www.nature.com/scientificreports/ emerged from E. heros eggs. Likewise, differences were also observed between female and male body lengths, and female body length (1.18 mm) was greater than male body length (1.11 mm) (F sex = 12.38; p sex = 0.0012) ( Table 2). Trissolcus urichi wing length (F host = 15.35; p host = 0.0004) and wing width (F host = 4.78; p host = 0.0354) also differed between the hosts. Trissolcus urichi emerged from E. heros eggs had a greater wing length (1.19 mm) than those from D. melacanthus eggs (1.10 mm). The wing length (F sex = 0.00; p sex = 0.9491) and wing width (F sex = 0.01; p sex = 0.9205 respectively) did not differ between sexes. In relation to the width of the wings, there was a difference between the hosts (F host = 4.78; p host = 0.0354) with larger values for the parasitoids that emerged from eggs of E. heros (0.42 mm) ( Table 2).

Discussion
The data presented here will contribute to understand life history of T. urichi and its most important biological traits regarding its parasitism on E. heros and D. melacanthus eggs. Such previous information is of theoretical and practical interest in order to later use this biocontrol agent to manage these species of stink bugs. Most of the published studies of parasitoids from the Scelionidae family report differences in their ability to parasitize depending upon the host species 15,[30][31][32] . Overall, T. urichi had preference to parasitize E. heros eggs over D. melacanthus eggs. This had been previously recorded for other scelionids 19,33 , however, as far as we know this is the first report for T. urichi.
Trissolcus urichi parasitism preference for E. heros over D. melacanthus eggs might be attributed to preimaginal learning during larval development 34,35 . Thus, adults of T. urichi would have preferred to parasitize eggs of E. heros eggs because they had been previously reared on this host species. Learning might also occur in young adults 36 . Often, changes in adult behavior are induced by chemical contamination carried over from the larval to the adult environment, known as 'chemical legacy' 37 , which occurrence can not be excluded from our trials. Furthermore, we might also speculate that T. urichi preference to parasitize E. heros eggs over D. melacanthus eggs observed in this study can be due to the ability of adult wasps to identify the best host, maximizing their reproductive success 38 . Different previous studies support this hypothesis, reporting that, given an abundance of hosts, parasitoids tend to avoid parasitism in hosts that present inferior nutritional qualities 39,40 . Thus, the possible better nutritional qualities of E. heros eggs over D. melacanthus eggs could be a plausible explanation to the higher performance of T. urichi in E. heros eggs, which exhibited higher number of parasitized eggs, greater emergence rate, and shorter time of egg-adult development, as well as the development of larger parasitoid adults (greater body length and greater wing length and width) when compared to results from D. melacanthus eggs. Even though, better nutritional value is frequently related to host size, this might not be applied to our results since E. heros eggs (0.83 mm width × 0.91 mm length) are smaller than D. melacanthus eggs (0.82 mm width × 0.98 mm length) 25 . Therefore, it is important to mention that host quality can vary not only with egg size but also with other factors such as host species 40 . Moreover, not only is nutritional quality of a host related to the physical but also chemical characteristics of each species 41,42 . Host chemical substances is known to have influence on parasitism 43 , which was not evaluated in this research. Future researches on this subject should also analyze host chemical composition of the studied hosts.
Egg-adult period (days) may indicate the quality of a specific host. The extended duration of the larva-adult period observed for T. urichi on D. melacanthus eggs reinforces the hypothesis that D. melacanthus eggs might be a worse nutritional host for the parasitoid. In the literature, longer larval period is described as a compensatory action to allow larvae feeding on a lower-quality host to achieve sufficient mass in order to pupate and successfully reach the adult stage 39,44 . In general, the development of insects depends on the quality of the food consumed in the juvenile stages, which may vary according to the host eggs 44 . More suitable hosts generally facilitate more rapid development of the larval phase of the parasitoid as observed for E. heros eggs 30 . Shorter egg-adult period can be considered a positive parasitoid feature to ABC programs, since it allows a greater number of parasitoid generations in the same time period, maximizing its control potential in the field 45 . Differences in host eggs had been previously described as an important feature for other parasitoid species (Trichogramma sp.) 46,47 . Different egg characteristics including surface and chorion structure, as well as changes in color during embryonic development and volume, differ between host species and may influence egg parasitism. All these peculiarities of each host species, as well as their relative differences, can affect not only T. urichi handling time and exploitation but also host suitability for parasitoid development, which also influences developmental time 46 .
Sex ratio is another important biological characteristic in ABC programs. The higher the proportion of females the better since they are responsible for parasitism 45 . It would had been expected that host quality would affect the sex ratio of progeny [48][49][50] . However, no difference in sex ratio was observed between the two hosts evaluated in this study, and both host species exhibited high proportion of females as it is desirable in ABC programs. It is important to mention that sex ratio recorded in our study was even higher than reported values from literature (0.49) for T. urichi parasitizing D. melacanthus eggs 15 . Therefore, it suggests that even though host quality differences between E. heros and D. melacanthus to T. urichi might exist, those differences are probably not sufficient to impact parasitoid sex ratio in T. urichi progeny.
Regarding the parasitoid morphometric characters evaluated in this study, it is important to mention that they are cited in the literature as good indicators of the quality of different hosts 25,51 . Therefore, the greater body length and wing length and width observed in T. urichi emerging from E. heros eggs ratifies the better nutritional conditions offered by this host than those offered by D. melacanthus eggs previously discussed for the comparative Scientific RepoRtS | (2020) 10:12441 | https://doi.org/10.1038/s41598-020-69406-z www.nature.com/scientificreports/ biology of the parasitoid in these hosts when T. urichi also presented greater parasitism and greater emergence of adults in E. heros eggs, in addition to the shorter time of egg-adult development. When referring to the dimensions of T. urichi it is important to note that the female body length is greater than the male. However, there were no differences between the length and width of the wing or length of the tibia. It was observed that the size difference between males and females tends to be greater in a smaller host 52 . As only the body length of T. urichi varied between males and females, this may indicate that despite the apparent superiority of E. heros as host of T. urichi, this parasitoid still exhibits good development in both hosts. Overall, we can conclude that T. urichi had better performance (not only higher parasitism and emergence but also parasitism preference and bigger parasitoid progeny) on E. heros eggs compared to D. melacanthus eggs, although the parasitoid had also acceptable parasitism capacity and development in D. melacanthus. Furthermore, as mentioned earlier, this information can also be used to predict T. urichi dynamics when used in biological control of stink bugs in integrated pest management.