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.

Introduction

Stink bugs are one of the most important groups of insects that cause yield losses in soybean (Glycine max) production in Brazil, Argentina and Uruguay[1]. Not only have they damage soybean in South America but also in Arkansas and other states in the Mid-South of the United States[2]. Because these insects feed directly on the soybean pods they seriously affect both yield and bean quality[1,3]. Among the recorded stink bugs from soybean fields, there have been at least 54 different species belonging to the family Pentatomidae[4]. The relative economic importance of each species might vary according to each country or its region[5]. However, of the many species of stink bug, the Neotropical Brown Stink Bug, Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) is the most frequent pest of field crops, mainly in the central region of Brazil at latitudes between 0° and 23°[6]. In addition, more recently, the Green-Belly Stink Bug, Dichelops melacanthus (Dallas) (Hemiptera: Pentatomidae), has become more abundant, increasing its significance to soybean and maize (Zea mays) production in the Neotropical region, especially in Brazil[7]. This increase is mostly a consequence of the adopted production system in which soybean is cropped during summer immediately followed by maize in autumn. The resulting continuous food supply to the insects throughout the year, known as green bridge, has favored D. melacanthus outbreaks[8].

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[10-12]. 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].

Egg parasitoids have been widely used in ABC and can be considered the most important stink bug biocontrol agent[14,15]. Among the egg parasitoid species, Telenomus podisi Ashmead (Hymenoptera: Scelionidae) is the most abundant and studied species[16-18]. However, despite being less studied, Trissolcus urichi (Crawford) (Hymenoptera: Scelionidae) is also among the most common egg parasitoids of stink bugs found in the Neotropical region[16,19,20]. This parasitoid species has been recorded among the most important South America soybean producers such as Brazil, Argentina, Uruguay and Paraguay as well as other countries of the Neotropical region (Mexico, Trinidad, Dominican Republic, Panama, Saint Kitts, Saint Lucia, Saint Vincent Island, Antigua and Barbados, Guyana and Bolivia) parasitizing eggs not only of E. heros and D. melacanthus, evaluated in this study but also eggs of Acrosternum aseadum Rolston, Antiteuchus variolosus Westwood, Brontocoris nigrolimbatus (Spinola), Dichelops furcatus (Fabricius), Edessa meditabunda Fabricius, Edessa rufomarginata (De Geer), Edessa spp., Nezara viridula (Linneo), Piezodorus guildinii (Westwood), Tibraca limbativentris Stal and Thyanta perditor (Fabricius) (Hemiptera, Pentatomidae), and Sphyrocoris obliquus (Hemiptera, Scutelleridae)[21].

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.

Material and methods

Laboratory rearing of T. urichi, E. heros, and D. melacanthus

Trissolcus urichi females as well as the studied hosts, E. heros and D. melacanthus, originated from insect colonies kept at Embrapa Soybean (one of the units of the Brazilian Agricultural Research Corporation), Londrina, State of Paraná, Brazil. Colonies were kept under controlled environmental conditions inside Biochemical Oxygen Demand (BOD) climate chambers (ELETROLab®, model EL 212, São Paulo, SP, Brazil) set at 80 ± 10% humidity, temperature of 25 ± 2 °C, and a 14:10 h (L:D) photoperiod according to methodologies previously described in literature[22,23] and briefly summarized in the followings.

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.

Bioassays

Three bioassays were conducted on E. heros and D. melacanthus eggs as follows: Trissolcus urichi host preference between eggs of E. heros and D. melacanthus (bioassay 1); Parasitism [egg-adult period (days), number of eggs parasitized in 24 h, emergence rate (%), and sex ratio] of E. heros and D. melacanthus eggs by T. urichi (bioassay 2); Trissolcus urichi adult morphometry when reared on E. heros and D. melacanthus eggs (bioassay 3). All trials were carried out in controlled environmental conditions inside BOD chambers (ELETROLab®, model EL 212, São Paulo, SP, Brazil) set at 25 ± 2 °C, relative humidity 80 ± 10%, and 14:10 h L:D photoperiod.

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].

Figure 1

Arenas used in the host preference test of the parasitoid Trissolcus urichi[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). Four mated T. urichi females (≤ 48 h old, mated with no previous parasitism experience) were released at the top of the arena (one female to every 40 eggs), for a period of 24 h, according to the methodology described in literature[24] for evaluation of host preference of Trichogramma parasitoids and later adapted[25] for evaluation of host preference in parasitoids of the genus Telenomus. A proportion of one parasitoid female was used for each 40 host eggs. After the 24-h period, cards were removed and kept in climatic chambers until emergence of adults. Preference for parasitism (%) for each host species was calculated following the equation: preference for parasitism (%) = number of eggs parasitized of each species/total number of parasitized eggs in the arena × 100. The number of parasitized eggs was calculated as the number of emerged parasitoids plus the number of adult parasitoids completely developed but dead inside the host (observed by means of dissections).

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.

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].

Results

Trissolcus urichi females clearly preferred E. heros eggs over D. melacanthus eggs for parasitism (F = 27.81; p ≤ 0.0001). The majority of parasitized eggs (63.15%) were E. heros eggs and only 36.85% were D. melacanthus eggs (Table 1).

Table 1

Preference of parasitism (bioassay 1) and biological characteristics (bioassay 2) of Trissolcus urichi on eggs of Euschistus heros and Dichelops melacanthus.

Host	Preference for parasitism (%)	Biological characteristics
		Egg-adult period (days)1	Eggs parasitized (n)1	Emergency (%)1	Sex ratio1
E. heros	63.15 ± 3.53a	13.15 ± 0.05b	16.15 ± 1.09a	93.41 ± 1.38a	0.78 ± 0.02 ns
D. melacanthus	36.85 ± 3.53b	14.30 ± 0.15a	11.63 ± 1.16b	82.84 ± 3.76b	0.72 ± 0.03
CV (%)	27.32	1.35	16.19	6.43	7.4
p	< 0.0001	0.0004	0.0293	0.0387	0.1758
F	27.81	66.68	8.1	6.96	2.35

1Means ± standard error (SE) followed by the same letter within a column, did not differ significantly (Tukey test, p ≤ 0,05). nsANOVA non-significant.

Trissolcus urichi egg-adult mean developmental time (days) was shorter for parasitoids reared on E. heros eggs (13.15 days) than for those reared on D. melacanthus eggs (14.30 days) (F = 66.68; p = 0.0004). The mean number of eggs parasitized by T. urichi was higher in E. heros (16.15 eggs or 40.38% parasitism) than in D. melacanthus (14.30 eggs or 35.75% parasitism) (F = 8.01; p = 0.0293). Similarly, parasitoid emergence (%) was higher for parasitoids reared on E. heros eggs (93.41%) (F = 6.96; p = 0.0387). The progeny sex ratio did not differ between the two hosts species (F = 2.35; p = 0.1758) (Table 1).

There was no interaction between host and sex in relation to T. urichi body length (Fhost*sex = 2.43; phost*sex = 0.1278); wing length (Fhost*sex = 1.63; phost*sex = 0.2097); wing width (Fhost*sex = 2.68; phost*sex = 0.1103) and tibia length (Fhost*sex = 0.10; phost*sex = 0.7519) (Table 2). Trissolcus urichi body length differed between the hosts (Fhost = 34.33; phost =  < 0.0001), with the greatest mean of body length (1.20 mm) observed for T. urichi that 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) (Fsex = 12.38; psex = 0.0012) (Table 2).

Table 2

Morphological characters (mm) of Trissolcus urichi reared on eggs of Euschistus heros and Dichelops melacanthus (bioassay 3).

Parameters	Morphological characters (mm)1
	Body length	Wing length	Wing Width	Tibia length
Host
E. heros	1.20 ± 0.02 a	1.19 ± 0.01 a	0.42 ± 0.01 a	0.34 ± 0.01 ns
D. melacanthus	1.09 ± 0.02 b	1.10 ± 0.02 b	0.39 ± 0.01 b	0.33 ± 0.01
Sex
Female	1.18 ± 0.02 A	1.14 ± 0.02 A	0.41 ± 0.01 A	0.33 ± 0.01
Male	1.11 ± 0.02 B	1.14 ± 0.02 B	0.41 ± 0.01 A	0.35 ± 0.01
Statistics
CV (%)	5.26	6.22	5.27	13.76
phost	< 0.0001	0.0004	0.0354	0.4853
psex	0.0012	0.9491	0.9205	0.1667
phost*sex	0.1278	0.2097	0.1103	0.7519
Fhost	34.33	15.35	4.78	0.50
Fsex	12.38	0.00	0.01	1.99
Fhost*sex	2.43	1.63	2.68	0.10

1Means ± EPM followed by the same letter in the column of each parameter (lowercase letters for host and capital letters for the genus of the parasitoid) do not differ significantly between each other (Tukey test, p ≤ 0,05). nsANOVA non-significant.

Trissolcus urichi wing length (Fhost = 15.35; phost = 0.0004) and wing width (Fhost = 4.78; phost = 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 (Fsex = 0.00; psex = 0.9491) and wing width (Fsex = 0.01; psex = 0.9205 respectively) did not differ between sexes. In relation to the width of the wings, there was a difference between the hosts (Fhost = 4.78; phost = 0.0354) with larger values for the parasitoids that emerged from eggs of E. heros (0.42 mm) (Table 2).

Contrary to previous reports, tibia length did not differ between hosts (Fhost = 0.50; phost = 0.4853), exhibiting similar length for E. heros (0.34 mm) and D. melacanthus (0.33 mm). Likewise, no differences were observed between female (0.33 mm) and male (0.35 mm) (Fsex = 1.99; psex = 0.1667) (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[15,30-32]. 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[48-50]. 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 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.