The sex ratio of the koinobiont parasitoid Microcharops anticarsiae Gupta remains female-biased on young larvae of velvetbean in the laboratory environment

The natural parasitic behavior of parasitoids should be known by those in charge of planning strategies for the biological control of pests; therefore, the aim of the present study was to determine the larval instar of Anticarsia gemmatalis Hübner parasitized by Microcharops anticarsiae Gupta in the field and the implication of such parasitic behavior in the sex ratio in the laboratory environment. The length of each larval instar of A. gemmatalis parasitized by M. anticarsiae in the field was determined, and the egg-to-pupa period of the parasitoid and its larval instar lengths were plotted in Gantt charts. According to the chart, A. gemmatalis was parasitized at the first (15%) and second (85%) larval instars in the field, but the length of the first, second and third larval instars of this species was not affected by the parasitism by M. anticarsiae in the field; however, its fourth larval instar was extended and the fifth one was shortened in 2015 but not affected in 2016. The sex ratio of A. gemmatalis larvae parasitized by M. anticarsiae in the field was female-biased, and the sex ratio of early parasitized larvae (3-day old) in the laboratory environment was also female-biased for three cultivated generations. The ‘generation’ factor has affected the egg-to-pupa, pupal and egg-to-adult periods of M. anticarsiae, since females pupated earlier than males in the egg-to-pupa period. Based on the results, M. anticarsiae mostly parasitized the second larval instar of A. gemmatalis in the field, and parasitism in 3-day old larvae in the laboratory environment produced female-biased sex ratio in M. anticarsiae, regardless of the generation.


Introduction
Successful parasitism in natural environment involves complex factors, such as choosing the right host (Ueno, 2015), number of ovipositions (Huang, Hua, Wang, Zhang, & Li, 2017), injecting venom to overcome the immune response of the host, and adapting or regulating the physiology of the parasitoid through larvae development (Kaeslin et al., 2010). According to Bernal, Prasifka, Sétamou, and Heinz (2004), parasitoids are the most documented examples of biological control worldwide, mainly species belonging to the order Hymenoptera. Studies on parasitoid wasps and their behavior are needed in order to help better understanding them and to use these species in agricultural biological control.
Microcharops anticarsiae Gupta (Hymenoptera: Ichneumonidae) is an efficient natural enemy of Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae), but also parasitizes other lepidopteran species (Patel & Habib, 1998). Although M. anticarsiae is a koinobiont parasite, adult females belonging to this species lay eggs inside the host. The parasitic larva feed upon the host from inside of it; however, the host continues to feed, grow and defend itself at least during the initial parasitism phase (Harvey, Poelman, & Tanaka, 2013).
Studies have determined that M. anticarsiae has four larval instars when parasitize A. gemmatalis larvae feeding on conventional (non-transgenic) soybean leaves. Microcharops anticarsiae parasitize the host from the first to fourth larval instar, but laboratory results showed no preference for a particular instar under laboratory conditions (Patel & Habib, 1993). Other studies have shown that parasitism on different instars did not have any effect on the larval development of M. anticarsiae; however, despite its potential to be a biological control agent in crops, the literature lacks studies focused on determining the parasitic behavior of parasitoids, such as M. anticarsiae, in the field and the implication of such parasitism on sex ratio in the laboratory environment.
The present study results from an investigation about the natural parasitic behavior of M. anticarsiae, variation in its sex ratio throughout three generations in the laboratory environment and its effect on the parasitoid development time.

Study Site
The experiments were conducted at the Teaching, Research and Extension Farm (21º15'04" S; 48º17'04" W) and at the Biological Control and Integrated Pest Management Laboratory of the Agricultural and Veterinarian Sciences School of Universidade Estadual Paulista "Júlio de Mesquita Filho", Jaboticabal Campus, State of São Paulo, Brazil (21º14'28" S; 48º17'23" W) during the 2015 and 2016 seasons. Samples were collected in January and February in both seasons. The Department of Exact Sciences of Universidade Estadual Paulista "Júlio de Mesquita Filho" provided the meteorological data used in the study.

Sample collection
Soybean cultivars SYN 1365 RR® and BRX Potência RR ® (both are tolerant to glyphosate) were sown in 1 ha during the 2015 and 2016 seasons, respectively -crops were not treated with insecticide during the experiment. Anticarsia gemmatalis larvae were collected with a beat sheet (length: 1 m -width: 0.5 m) in a crop row (length: 2 m) according to Souza et al. (2018); in total, 20 points were randomly sampled on a weekly basis after plant emergence. The collected larvae were stored in plastic containers and sent to the laboratory.

Sample monitoring in laboratory environment
The collected larvae were stored in the Biological Control and Integrated Pest Management Laboratory under controlled temperature (25 ± 2°C) and relative humidity (60 ± 10%) conditions, in a 14:8-h photoperiod. Larvae were grouped based on their field instar; and individually placed in Petri dishes. Leaflets, which were daily replaced, covered the bottom of the Petri dishes for larvae feeding. Soybean leaves were previously cleaned with sodium hypochlorite (2.5%) for five minutes and washed three times in water. Petri dishes were sealed with polyvinyl chloride film to avoid leaflet dehydration. It was not possible differentiating the parasitized larvae from those non-parasitized; therefore, it was only possible to separate the parasitized group from the non-parasitized one when parasitoid larvae left the hosts to form the cocoon. A brief description of the experimental groups is presented in Table 1. The following parameters were measured after larval collection in the field in the 2015 and 2016 seasons: Length of each larval instar, larval period of A. gemmatalis and the sex ratio of M. anticarsiae. Larval instars of A. gemmatalis grown in the laboratory environment were observed on a daily basis and changes on them were recorded at the same frequency. Larvae molt was characterized by the presence of shed head capsules in each Petri dish. Sex ratio was determined based on the ratio of females to the total population. Gantt charts of larval instar length and the parasitoid egg-to-pupa period were plotted to determine the larval instar of A. gemmatalis parasitized by M. anticarsiae in the field.
Based on the Gantt charts, larval instar lengths were organized in ascending order (from first to fifth instar). The egg-to-pupa period of M. anticarsiae parasitizing A. gemmatalis larvae was characterized by the first instar of larvae parasitized in the field, and this variable was used to determine the larval instar of A. gemmatalis parasitized by M. anticarsiae in the field. Laboratory culture was based on measurements of the following parameters: Egg-to-pupa, pupal and egg-to-adult periods.

Microcharops anticarsiae culture in laboratory environment
Microcharops anticarsiae emerged from larvae collected in the field in the 2016 season were used to start the culture in the laboratory environment. Adult specimens represented the parental generation of the three cultivated generations. In total, 10 larvae (3-day old) of A. gemmatalis were conditioned in plastic container (5 L) with one drop of pure honey and a coffee cup (10 mL) of wet cotton; the container had two lateral openings (10 x 5 cm) covered with a mesh for airflow purposes. A glass pot (10 mL) was used to hold the soybean leaf. Larvae were placed on soybean leaves before parasitism began. Microcharops anticarsiae females were mated at the age of 8 days, prior to parasitism. One mated female was placed in the experimental container, and stayed there for 2 hours; this procedure was repeated 10 times, but with different females and larvae in each repetition. Larvae were placed in Petri dishes in separate, and fed soybean leaflet, similar to the procedure adopted for larvae collected in the field. All parasitoid females were released at Jaboticabal Campus after parasitism. Microcharops anticarsiae males and females fed on pure honey and wet cotton.

Statistical analysis
Statistical analyses were carried out in the R software for Windows (R Core Team, 2016). Normal ity was checked by the Shapiro-Wilk test and the Levene test was used to evaluate homogeneity of variances (α ≤ 0.05). Data were tested by one-way ANOVA, followed by Duncan's test for comparison between factors. Differences were significant at α ≤ 0.05. Data were transformed into y λ by Box-Cox transformation, whenever necessary. Data of the laboratory culture were analyzed by repeated measures ANOVA.

Ethical note
Parasitism condition and the sex of larvae collected in the field were unknown at sampling. Parasitoids emerged from larvae collected in the field were fed on water and honey (10%), and were used in laboratory studies. Sample collection in the field was carried out based on the Brazilian legislation for scientific activities. The first author was granted with Authorization 49516 -1 to carry out the research between 2014 and 2016. Adult parasitoids were sent to the American Entomological Institute for identification and those identified were deposited in the Museum of Entomology of Universidade Estadual Paulista "Júlio de Mesquita Filho" at the Agricultural and Veterinarian Sciences School, Jaboticabal Campus, State of São Paulo, Brazil.

Results
Larval development of A. gemmatalis parasitized with M. anticarsiae in the field Based on results of the 2015 season, there were no differences in length for the first, second and third larval instars of A. gemmatalis parasitized by M. anticarsiae in the field (Table 2). However, the length of the fourth larval instar was extended by parasitism, in turn, the length of the fifth larval instar was shortened; consequently, the larval period was in general shorter (Tables 2 and 3). The egg-to-pupa period (11.07 ± 0.46 days) of M. anticarsiae parasitizing A. gemmatalis was determined based on A. gemmatalis larvae collected in the field at the first instar (G1p).
The natural infestation of A. gemmatalis was low in 2016; therefore, there were only few larvae to be sampled in the field and to form the experimental groups (Table 4 and 5). Based on the results, there were no differences in the length of the third larval instar of A. gemmatalis parasitized by M. anticarsiae in the field (G3 vs G3p), but the length of the fourth larval instar of this species was extended due to parasitism (G3 vs G3p and G4 vs G4p), and the length of the fifth larval instar was not affected in the field (G4 vs G4p). The total larval period of A. gemmatalis was shorter.
Some relevant factors about parasitism by M. anticarsiae were observed: its larvae ate A. gemmatalis larvae from inside of it and at the end of the development period, only the integument and the head of the hosts remained intact. Microcharops anticarsiae larvae wove a cocoon and pupated on the sides of their host. M. anticarsiae males were ready to copulate right after their emergence, but females were not.

Determination of A. gemmatalis larval instar parasitized by M. anticarsiae in the field
The egg-to-pupa period (11.07 ± 0.46 days) was assessed through Gantt chart plots and results were used to determine the larval instar parasitized by M. anticarsiae in the field in the 2015 season (Table  2). Considering Figure 1, A. gemmatalis larvae in G1p were parasitized at their first larval instar and larvae at G2p, G3p and G4p were parasitized at their second instar, as shown in the Gantt chart. In total, 79 larvae from groups G2p (N = 17), G3p (N = 28) and G4p (N = 34) were parasitized by M. anticarsiae at their second larval instar in the field (Table 2). In addition, 14 larvae of A. gemmatalis from G1p were parasitized at their first larval instar; therefore, the host larvae were parasitized at the first (15%) and second larval instars (85%) in the 2015 season. Unfortunately, it was not possible to arrange groups with individuals collected in the 2016 season, given the low natural infestation with A. gemmatalis in the field (Tables 4 and 5).

Sex ratio and development time of M. anticarsiae parasitizing A. gemmatalis
The sex ratio of M. anticarsiae was female-biased in the 2015 season, except for G3p, which recorded sex ratio of 0.49 (Table 2). Based on the observed sex ratio in the 2016 season, the population of M. anticarsiae was composed of 50% females, except for G2p, which was female-biased (Tables 4 and 5). Adult M. anticarsiae emerged in the 2016 season were used to start the laboratory culture, based on the preliminary results of the 2015 season.
The sex ratios of the first, second and third cultured generations of M. anticarsiae parasitizing 3-day old A. gemmatalis larvae were 0.56, 0.48 and 0.58, respectively; therefore, parasitism rate in these generations reached 92%, 81% and 90%, respectively ( Table 6). The sex ratio of M. anticarsiae was female-biased when A. gemmatalis larvae were parasitized at the age of 3 days.  The 'generation' factor affected the egg-to-pupa, pupal and egg-to-adult periods, and the factor 'sex' affected the egg-to-pupa period of M. anticarsiae parasitizing A. gemmatalis (Table 7). The egg-to-pupa, pupal and egg-to-adult periods were longer in the third generation of M. anticarsiae, and the egg-to-pupa period was shorter in females than in males (Figure 2). anticarsiae.

Discussion
Larval development of A. gemmatalis parasitized by M. anticarsiae in the field Parasitism by M. anticarsiae shortened the length of the fourth larval instar of A. gemmatalis and extended the fifth instar, but the overall larval period was extended. The effects of M. anticarsiae parasitism on the length of larval instars of parasitized A. gemmatalis larvae is explained by changes in growth hormone regulation, which resulted from the development of immature parasitoids (Kaeslin et al., 2010).
Larval period shortening was a positive outcome, because it shortened the time that parasitized A. gemmatalis larvae require to feed on soybean leaves. Therefore, parasitized A. gemmatalis larvae had shorter time to cause damage to soybean plants in the field.
Parasitoid larvae left the host and pupated on the sides of it. Assumingly, immature parasitoids allow their host larvae to develop to a certain size (mostly, to the fourth instar, when these host larvae meet their nutritional requirements) before they finally start eating the host from inside of it. A few parasitoid larvae left the host at the fifth larval instar; this finding can be likely explained by the higher cost associated with overcoming its immune defenses, with development control, and with the growth arrest of larger hosts (Kaeslin et al., 2010;Zhou, Meng, & Li, 2017).

Determination of A. gemmatalis larval instars parasitized by M. anticarsiae in the field
The egg-to-pupa period of M. anticarsiae in group G1p was used to determine the larval instar of parasitized A. gemmatalis larvae. This development time did not change when A. gemmatalis was parasitized at its first, second, third or fourth larval instars. This phenomenon occurs in other endoparasitoids; for instance, the development time of Microplitis mediator (Haliday) (Hymenoptera: Braconidae) is not affected by parasitizing host larvae of Mamestra brassicae (Linnaeus) (Lepidoptera: Noctuidae) at the first, second or third instar (Malcicka & Harvey, 2014), which indicates that endoparasitoids strategically maintain the development time changing other parameters like weight and sex ratio. Therefore, we considered the egg-topupa period needed by M. anticarsiae at the first instar (G1p) of A. gemmatalis was not different from that needed by M. anticarsiae to parasitize the second, third, and fourth larval instars of the assessed wasp species.
Based on Figure 1, M. anticarsiae parasitized the first and second larval instars of A. gemmatalis in the field; therefore, it is possible to state that M. anticarsiae more often parasitizes the early instars of A. gemmatalis in order to minimize mortality risks inherent in parasitism in older instars. Older larvae of A. gemmatalis are more efficient in attacking and harming the parasitoid during the parasitism process (Ameri, Rasekh, & Michaud, 2014;Firlej, Lucas, Coderre, & Boivin, 2010;Zhou et al., 2017); furthermore, parasitoid larvae have more time to develop better physiological conditions inside young hosts, since these hosts have less effective immune responses and/or produce fewer immune defenses than larger and more developed hosts (Kaeslin et al., 2010).
The present study sheds light on unanswered questions about the parasitic behavior of M. anticarsiae in the field, since the recorded findings provide important information about large-scale culture production methodologies focused on growing biological control agents to be applied to pests in crops.

Sex ratio and development time of M. anticarsiae parasitizing A. gemmatalis
Microcharops anticarsiae offspring was female-biased in most groups. The sex ratio is a critical factor to the mitigation of costs with the culture of this parasitoid, as well as an important attribute of biological control agents (Pandey, Kumar, & Tripathi, 2004). According to the literature, the sex ratio of Hymenopteran is influenced by factors such as diet (Ongaratto et al., 2019), instar (Poncio et al., 2018;Chu et al., 2014), superparasitism (Alvarenga, Dias, Stuhl, & Sivinski, 2015) and size/age of the host (Van Nieuwenhove & Ovruski, 2011;Ueno, 2015). Also, chemical cues are associated with the host characteristics, such as feces (Faraone, Svensson, & Anderbrant, 2017) and its ability to feed on different host plants (Morawo & Fadamiro, 2019). According to the present results, female M. anticarsiae individuals parasitize A. gemmatalis larvae at the end of the first and at the beginning of the second instar; these are the most favorable periods for parasitism for two reasons: larvae attach to the substrate at the end of the first instar and stop feeding, and the larvae stops moving at this point, M. anticarsiae has the advantage in parasitizing immobilized larvae; second reason, larvae remain weak at the beginning of the second instar and are easily exposed to parasitism -parasitoids can take advantage of this weak condition. Assumingly, M. anticarsiae can detect the volatiles emitted by early A. gemmatalis larvae (first and second instars) at molting; therefore, the end of the first and the beginning of the second instar may be the most favorable periods to parasitism, because they enable M. anticarsiae to produce female-biased sex ratio.
The effect of the factor 'generation' on the development time of M. anticarsiae indicates that the laboratory culture procedure needs improvements in order to avoid negative effects on the parasitoid development as reported by Bueno, Romero, Osorio, and Zaviezo (2017) for Mastrus ridens Horstmann (Hymenoptera: Ichneumonidae), because there were no introduced new parasitoids to the laboratory culture due to the lack of larvae in soybean crops in the field and laboratory. Using wind tunnels to help parasitoids finding the hosts, as well as olfactometers to determine the age of females to mate, could be instruments used to achieve such improvements. Otherwise, consanguinity had negative influence on the development time of third-generation parasitoids, because there were no new parasitoids in the laboratory culture due to the lack of larvae in soybean crops in the field. The difference between the egg-to-pupa period of males and females helps separating parasitoids by sex, even before emergence. The rejection of recently emerged M. anticarsiae females indicates that they rather feed or develop before mating. Our results provide important information to those coordinating the production of M. anticarsiae females in large-scale culture production programs focused on biological control.
The current study is expected to help and motivate new researchers to conduct other investigations on this topic, since there are more questions to be answered about parasitic behavior and host-parasitoid interactions in the field. This research broadens the knowledge about the development of biological control programs focused on agricultural applications.

Conclusion
Parasitism of M. anticarsiae is female-biased in 3-day old A. gemmatalis, in the laboratory environment. The egg-to-pupa, pupal, and egg-to-adult periods of M. anticarsiae parasitizing 3-day old A. gemmatalis were affected only in the third generation when there is no introduction of new individuals in the culture. The egg-to-pupa period is shorter in females than in males of M. anticarsiae.