The experiments were conducted during the maize-growing season (May–November 2022) under controlled laboratory conditions (mean temperature 24.3 ± 0.8 °C and RH 69.3 ± 2.2%) at the Entomology Laboratory of the National Maize Research Program (NMRP), Rampur, Chitwan, Nepal (27°39′17″N, 84°21′2″E; 228 m above sea level).

Late-instar S. frugiperda larvae were collected from pesticide-free maize fields at NMRP and reared on fresh maize leaves in transparent plastic containers (19 x 14 x 7 cm³) for cohort-based rearing (33). Fresh green leaves were provided every one to two days, depending on their condition, until the larvae pupated. The pupae were collected daily and transferred to separate boxes (14 × 10 × 4 cm³). This rearing process was repeated to ensure a continuous supply of pupae for adult emergence.

The adults after emergence were transferred to oviposition cages (30 × 30 × 30 cm3) supplied with water-soaked cotton, honey, and young maize plants for egg laying. The next morning, maize plants with eggs on their leaves were carefully removed. Leaves with egg masses were clipped and placed in a well-ventilated box. Medium-sized multilayered egg masses (roughly 100 eggs) were then selected and categorized into three groups based on scale covering: fully covered, partially covered, and uncovered. Similarly, single-layer egg masses were counted under a microscope using a fine camel hairbrush, and batches of 20, 40, and 60 eggs were prepared. This process was repeated every day to obtain eggs of different ages for each group. Finally, the eggs were glued onto cardboard pieces (3 × 5 cm²) to prepare standardized egg cards for the experiment.

A wild population of T. remus were collected from the NMRP maize field and a nucleus colony was established in the laboratory. FAW egg masses were randomly collected and checked for the presence of parasitoids. The unparasitized eggs yields only FAW larvae whereas, parasitized eggs produced few or no FAW larvae after 1–3 days and adult parasitoids emerged after about 7–10 days. The emerged parasitoids were supplied with a thin layer of honey as source of energy and later identified to confirm the species. To multiply them, fresh FAW egg masses (from egg collection chamber) were irradiated in a UV chamber for 15–20 minutes to halt the development of embryo. The egg cards were then exposed to the test tube containing parasitoids at a ratio of one parasitoid per 20 eggs for 24 hours, with fresh FAW eggs supplied on alternate days until the parasitoids died, as described by Tefera et al. (2019) (14). The parasitized egg masses were kept in separate test tube, and the process was repeated for 3–4 generations. The parasitized egg masses (master’s card) were prepared and refrigerated at 4–6 °C for 1–2 days at parasitoid pupal stage. These master cards were then taken out each day to make sure that freshly emerged adult parasitoids are available for research every day.

The other species of egg parasitoid, T. chilonis was being successfully reared in the entomological division of NMRP, Rampur. Tricho-master cards (card with glued eggs of Corcyra cephalonica (Stainton, 1866) already parasitized by T. chilonis) were used to parasitize few fresh batches of FAW eggs. The parasitized egg masses were then used to parasitized and multiply other batches of eggs as explained for T. remus and after 3–4 generations, multiple batches were made and refrigerated to ensure a continuous supply of new parasitoid every day in the laboratory.

In nature, egg parasitoids come across FAW eggs in different numbers and at different ages. The number of eggs depends on the age of the female FAW—newly mated females lay many eggs, while older ones lay fewer. FAW eggs also hatch in about 3 days, so their age varies depending on the oviposition time. To study how these differences in egg number and age affect parasitism, two egg parasitoids—T. chilonis and T. remus—were tested on FAW eggs of three different densities (20, 40, and 60 eggs) and three age groups (less than 12 hours, 24–36 hours, and 48–60 hours old). The experiment was set up using a completely randomized design (CRD) with three replications. For each test, a one-day-old mated female parasitoid was placed inside a glass test tube (15 mm × 100 mm). Data were collected on the proportion of parasitism, viable adult emergence, sex ratio (percentage of female progeny), and the developmental period of both parasitoid species.

Newly mated female lays egg mass with fully covered scale and, the proportion of scale decreases and old female lays eggs without scale covering. To study how these differences in egg scale covering and age affect parasitism, T. chilonis and T. remus were evaluated on FAW eggs of three different scale covering (Fully covered, partially covered, and uncovered) and three age groups (less than 12 hours, 24–36 hours, and 48–60 hours old). The experiment was also set up using a completely randomized design (CRD) with three replications. Uniform sized egg masses (nearly 100 ± 10) were kept in a test tube and a one-day-old mated female parasitoid was placed inside a glass test tube (15 mm × 100 mm). Data were collected on the proportion of parasitism, viable adult emergence, sex ratio (percentage of female progeny), and the developmental period of both parasitoid species.

All data were entered into MS Excel (Office 365) for basic analysis and graph generation. Normality and homogeneity were tested using the GVLMA package, and data not meeting ANOVA assumptions were arcsine-transformed. ANOVA was conducted in RStudio (v2022.03.1), and significant results were separated using Tukey’s HSD at a 5% significance level. Univariate outliers present on the datasets were identified using box and whisker plot techniques. The influential outliers compromising and questioning the credibility of the result were handled by the variable deletion techniques i.e., data exclusion as explained by Mowbray et al. (2019) (34). Data on percentage emergence of parasitoids, female progeny percentage and developmental period for experiment 2 were compromised because of the undefined value of T. chilonis on one variable (fully covered). The variable was excluded for the unbiased estimate of those parameters.

This study aimed to evaluate the parasitism efficiency of T. remus and T. chilonis on S. frugiperda egg masses of varying structures and ages. Our finding demonstrated that T. remus exhibited significantly higher parasitism in both single and multilayered egg masses. Parasitism was higher for T. remus than T. chilonis in both single and multilayered egg masses, with the highest rates in single layered egg mass due to easier accessibility for parasitoids (28). The parasitism declines with egg mass layers and scale covering, reflecting the physical challenge of overcoming scales and penetrating deeper layers. These findings are supported by Beserra and Parra (2005) (28), Jin et al. (2021) (29), Mohamed (2021) (35) and Mohamed et al. (2023) (36), all of them confirmed the parasitism rates by different Trichogramma species decreased based on the number of egg mass layer.

The superior performance of T. remus is largely due to its robust and aggressive nature enabling it to overcome the physical barrier of scale coverings and penetrate deeper into multilayered egg masses (13). In contrast, T. chilonis was hindered by the scale covering, restricting it to parasitize only the upper layers or eggs on the edges (24, 30).

Parasitism decreased with increasing host egg age due to depletion of nutrient and host embryo development, which rendered older eggs unsuitable for parasitoid development (31). Also, the egg scale covering harden with age, making it more challenging for parasitoids to penetrate through it (37). Although these results demonstrate T. remus’s superiority over T. chilonis as a biocontrol agent, field complexities may not be fully captured by laboratory conditions. Future studies should evaluate their parasitic performance under natural conditions and investigate combined control methods that use both parasitoids.

This study investigated the emergence success of T. remus and T. chilonis from S. frugiperda egg masses of varying structures and ages. The results revealed significantly higher adult emergence percentage for T. remus compared to T. chilonis, which may be due to its stronger adaptation to S. frugiperda eggs. These findings are supported by Carneiro and Fernandes (2012) (24), who also documented higher emergence rate of T. remus than T. pretiosum on similar host systems.

Parasitoids emergence is influenced by host egg age. As host egg age increased, emergence rates declined, indicating that older eggs likely became less suitable for parasitoid development. This is likely due to nutrient depletion and progressive host embryo development in older eggs, which fail to support parasitoid growth and lead to higher mortality before emergence —a pattern also observed by Tuncbilek and Ayvaz (2003) (31). The findings are also supported by Priyanka et al. (2023) (32), who reported a decrease in parasitoid emergence with increasing host egg age. The emergence rates of both species were not significantly impacted by egg scale covering, suggesting this physical barrier does not hinder larval development after oviposition. This finding is supported by Laminou et al. (2020) (13) and Mohamed et al. (2023), who reported no effects of FAW egg scale coverage on emergence of egg parasitoids. These results imply that fresh or young host eggs are suitable for mass rearing of parasitoids. Additionally, T. remus seems to be a good option for field release against S. frugiperda, particularly in cases where host egg age varies. Future research should validate these findings in the field and look into possible explanations for the developmental advantages of T. remus offspring over those of other species.

This study investigated the influence of host egg density, egg scale covering and egg age on the percentage of female progeny in two egg parasitoids, T. chilonis and T. remus. Our study reveals that the percentage of female progeny in both species of egg parasitoids are statistically similar. This finding is in consistent with the finding of Jin et al. (2021) (29) and Chen et al. (2021) (38), who too reported the average percentage of female progeny in T. chilonis and T. remus around 70 percent.

The proportion of female offspring decreased with increasing host egg densities. This might be explained by the fact that more unfertilized S. frugiperda eggs were parasitized in high-density environments, which probably produced more male progeny (29). Additionally, the percentage of female progeny declined with increasing egg age. This may be possibly due to declining egg quality and reduced resources in older eggs, which negatively impact the development of female parasitoids. This is supported by findings from Sun et al. (2021) (30), who observed a drop in Trichogramma female progeny percentage from 82% to 69% as egg age increased from 0 to 2 days. Similarly, Priyanka et al. (2023) (32) noted a reduction in T. remus female progeny from 66% to 64% as egg age increased from 24 to 48 hours. Our results demonstrate that the sex ratio of emerging parasitoids is significantly influenced by the age and density of the host egg. Because more females can increase the success of parasitism, this is beneficial for mass-rearing and biocontrol initiatives. These effects should be tested in other host-parasitoid systems and in the field.

This study investigated the influence of host egg density, egg scale covering and egg age on the developmental duration of two egg parasitoids, T. chilonis and T. remus. Our finding reveals that the developmental time from egg to adult remained consistent across both T. chilonis and T. remus, regardless of treatment conditions. This is supported by findings of Sultan et al. (2013) (39) who reported a development period of 9.60 days for T. chilonis, while Oktaviani et al. (2021) (40), observed a similar duration of 9.61 days for T. remus.

The reason behind this stability in developmental time may lie in the biology of the parasitoids. As noted by Bueno et al. (2008) (41) these parasitoid species lay their eggs singly inside individual FAW eggs and superparasitism or multiple parasitism are rare (42). Therefore, each parasitoid larva develops independently within a single host egg, external factors such as egg density or the presence of scale covering do not appear to influence their development. Moreover, our findings are consistent with Priyanka et al. (2023) (32), who recorded developmental durations of T. remus as 9.61 days on 24-hour-old eggs and 9.52 days on 48-hour-old eggs. These results further support the conclusion that the age of the host egg does not significantly impact the time required for parasitoid development. Although the developmental duration of these two parasitoids is consistent across different egg traits, the small sample size and laboratory setting necessitate larger field-based research to validate and generalize the findings.