Reproductive Success of Trichogramma ostriniae over Trichogramma dendrolimi in Multi-Generational Rearing on Corn Borer Eggs

Simple Summary

To evaluate reproductive success, the emergence of adult offsprings of two egg parasitoids, Trichogramma dendrolimi Matsumura and T. ostriniae Pang and Chen from the host eggs (Asian corn borer, Ostrinia furnacalis Guenée), as well as the adult offspring mortality from the unhatched host eggs, was compared under different parasitoid ratios across multiple generations. We discovered that both Trichogramma species’ offspring emergence were significantly influenced by the parasitoid generations, parasitoid ratios, and their interactions. The offspring mortality in both Trichogramma species was significantly affected by parasitoid generations but was not significantly influenced by parasitoid ratios or interaction between parasitoid generations and parasitoid ratios. Furthermore, across all parasitoid ratios, T. ostriniae outcompeted T. dendrolimi by F₃ generation, achieving full emergence while completely suppressing T. dendrolimi emergence. Moreover, after assessing the offspring mortality in our research by dissecting the unhatched eggs, we found that, across all parasitoid ratios and generations, the offspring mortality of T. ostriniae was considerably greater than that of T. dendrolimi. These results suggest that mortality is a crucial empirical measure that validates T. ostriniae’s superiority over T. dendrolimi.

Abstract

In China, the Asian corn borer (ACB), Ostrinia furnacalis (Guenee) (Lepidoptera: Pyralidae), is the most significant economic insect pest of corn, causing losses ranging from six to nine million tons annually by feeding on all parts of maize, including damaging ears and leaves and making tunnels in stems. In China, since the 1970s, the Trichogramma species have extensively mass-reared from factitious hosts to control ACB and support integrated pest management programs. The Trichogramma dendrolimi Matsumura and T. ostriniae Pang and Chen are the most efficient biocontrol agents for controlling ACB among the available Trichogramma species. To evaluate the reproductive success of Trichogramma dendrolimi and T. ostriniae, we assessed the impact of varying parasitoid ratios (5:1, 3:1, 1:1, 1:3, and 1:5 representing T. dendrolimi and T. ostriniae, respectively) on adult offspring emergence and mortality from ACB eggs over multiple generations (from first (F₁) to third (F₃) generations). We discovered that both Trichogramma species’ offspring emergence was significantly influenced by the parasitoid generations, parasitoid ratios, and their interactions. The offspring mortality in both Trichogramma species was significantly affected by parasitoid generations but was not significantly influenced by parasitoid ratios or interaction between parasitoid generations and parasitoid ratios. Furthermore, at parasitoid ratios of 5:1, 3:1, and 1:1, the emergence rate of the F₁ generation of T. dendrolimi was significantly higher compared to the ratios of 1:3 and 1:5. However, in the F₂ generation, the emergence of T. dendrolimi decreased considerably, and no emergence was observed in the F₃ generation. A contrasting trend was observed in the emergence of T. ostriniae offspring. Overall, regardless of the parasitoid ratios, the offspring emergence of T. ostriniae in all three generations was significantly higher than that of T. dendrolimi. After assessing the offspring mortality in our research by dissecting the unhatched eggs, we found an inverse relationship between the T. dendrolimi generations and their mortality across different parasitoid ratios. Notably, mortality exhibited a significant decline with an increasing number of generations. A positive correlation was observed between the number of T. ostriniae generations and their mortality across different parasitoid ratios, indicating that mortality increased with successive generations. Overall, across all parasitoid ratios and generations, the offspring mortality of T. ostriniae was considerably greater than that of T. dendrolimi. These results suggest that mortality is a crucial empirical measure that validates T. ostriniae’s superiority over T. dendrolimi. These findings highlight the importance of selecting suitable parasitoid species when implementing Trichogramma for pest management.

1. Introduction

For more than sixty years, the cultivated corn in the Western Pacific region of Asia was prone to the highly devastating Asian corn borer (ACB), Ostrinia furnacalis (Guenee) (Lepidoptera: Pyralidae) [1,2,3]. The distribution of the ACB extends northward across Northern China and eastward from India to Southern China, Japan, Korea, and the Philippines, reaching as far as Australia and the Solomon Islands [1,4,5,6,7]. The ACB larvae have caused more than thirty percent loss of maize products by feeding on all parts of maize, including damaging ears and leaves and making tunnels in stems [1,8,9]. Globally, maize is the leader among grain crops in terms of the highest planting areas [10]. In China, the ACB is the most significant economic insect pest of corn, causing losses ranging from six to nine million tons annually [11,12]. The synthetic insecticides serve as agents to control insect pests, such as ACB [13,14]. The extensive and indiscriminate application of synthetic chemicals negatively affect people’s health and natural environment [13]. Therefore, biological control is a deliberate, promising, and economical approach to managing insect pests worldwide [15].

The members of the family Trichogrammatidae are the most commonly used natural enemies for biological control programs [15]. Currently, Trichogrammatidae comprises about eight hundred species belonging to ninety genera [16]. The Trichogramma is the largest genus and contains about two hundred and thirty species [16]. The Trichogramma dendrolimi Matsumura and T. ostriniae Pang and Chen are the most efficient biocontrol agents for controlling ACB among the available Trichogramma species [15]. In China, since the 1970s, the Trichogramma species are extensively mass reared from factitious hosts to control ACB [17] and supporting integrated pest management programs [18]. Iqbal et al. [19] revealed that rearing T. ostriniae on the factitious host, Antheraea pernyi Guerin-Meneville, 1855, via multiparasitism with Trichogramma chilonis Ishii facilitates enhanced biocontrol potential against ACB. Furthermore, the effect of various parasitoid ratios on the numeric response of the Trichogramma species is indeed also crucial for optimizing their role in biological control programs. Ghaemmaghami et al. [20] observed the changes in functional and numerical responses of the parasitoid wasp, Trichogramma brassicae Bezdenko, 1968, over forty-five generations of rearing on Ephestia kuehniella Zeller, 1879. The parasitism and offspring’s emergence are the key functional and numerical responses that determine the fate of the effectiveness of parasitoid mass-rearing systems [20,21,22]. Regarding the importance of multiparasitism in parasitoid mass-rearing, Iqbal et al. [23] revealed that the optimal offspring emergence from one host egg (Chinese oak silkworm, Antheraea pernyi Guerin-Meneville) was observed when the T. ostriniae were in multiparasitism with T. chilonis. In China, T. chilonis, T. dendrolimi, and T. ostriniae are efficiently mass reproduced on A. pernyi through monoparasitism [24]. Therefore, more research is needed to explore the performance of T. ostriniae on ACB eggs and investigate the offspring’s emergence through multiparasitism along with T. dendrolimi.

Reproductive success plays a pivotal role in understanding the dynamics of multiparasitism in Trichogramma species, influencing both their competitive interactions and effectiveness in biological control [25]. Li et al. [26] revealed that T. dendrolimi showed reproductive success over T. ostriniae due to more adults emerging from A. pernyi eggs under a multiparasitism regime. Moreover, T. dendrolimi played a pivotal role in helping the optimal emergence of T. ostriniae offsprings because T. ostriniae offsprings failed to make enough holes in the host egg for their emergence due to the hard chorion of A. pernyi egg; therefore, T. ostriniae utilized the hole created by the T. dendrolimi for the exit [26]. More research is required to investigate the reproductive efficiency of these two parasitoid species at varying ratios while rearing on ACB eggs. Therefore, in the present study, we evaluated the offspring emergence and mortality of T. dendrolimi and T. ostriniae from ACB eggs at multiple generations under laboratory conditions to provide insights into using Trichogramma species for biological control of the Asian corn borer, making pest management more efficient, cost-effective, and environmentally sustainable.

2. Materials and Methods

2.1. Parasitoids

In 2023, the adult parasitoids T. dendrolimi and T. ostriniae were obtained from the parasitized ACB eggs in the corn fields of Changchun, Jilin province, China (43.89° N, 125.32° E). Based on the morphological characteristics of the male genitalia, both parasitoid species were identified using scanning electron microscope micrographs [27], and rDNA ITS2 sequence analysis confirmed this identification [28]. In the laboratory, these parasitoids were cultured on ACB eggs for five generations in an incubator (MLR-351H; Sanyo Corporation, Moriguchi, Osaka, Japan) under optimum conditions (L14: D10, 26 ± 1 °C, 65 ± 5% RH).

2.2. Host

Asian Corn Borer (ACB), Ostrinia furnacalis

ACB pupae were maintained in a cage (35 × 35 × 35 cm; Bugdorm-I, Taichung, Taiwan, China) at feasible conditions (L14: D10, 26 ± 1 °C, 65 ± 5% RH) to collect eggs for experimentation. After the emergence, the moths were provided with a 20% honey solution as a sustainable food source, and a large piece (30 cm × 30 cm) of wax paper was suspended in the cage for oviposition. After oviposition, the part of the wax paper containing egg masses was cut with a scissor and kept in a climate chamber room (Faithful Instrument Co., Ltd., Huanghua, Hebei, China) under optimal conditions (L14: D10, 26 ± 1 °C, 65 ± 5% RH) until they attained the age of less than 4 h for experimentation.

2.3. Comparative Reproductive Success of Trichogramma Species Across Generations

To assess the reproductive success between T. dendrolimi and T. ostriniae, the ACB eggs (aged < 4 h) were parasitized under controlled environmental conditions (L14: D10, 26 ± 1 °C, 65 ± 5% RH). For this purpose, the wax paper containing 70 ACB eggs was stapled to the dorsal leaf surface of a corn plant placed in a cage (2 × 2 × 2 m; Bugdorm-I, Taichung, Taiwan, China). Subsequently, each mated adult female of T. dendrolimi and T. ostriniae that had newly emerged (<12 h prior) was introduced into the cage containing ACB eggs for parasitization. Additionally, more diverse ratios of T. dendrolimi and T. ostriniae (3:1, 5:1, 1:3, and 1:5) were examined in four separate cages containing ACB eggs with a corn plant. After 8 days of parasitism, the parasitized eggs from all the cages were transferred to individual glass tubes (10 × 3 cm, length × diameter; Shanghai Allcan Medical Co., Ltd., Pudong New Area, Shanghai, China) and placed in an incubator under controlled conditions (L14: D10, 26 ± 1 °C, 65 ± 5% RH) for development. The emergence of the first generation (F₁) of both wasps was monitored daily, and cotton-soaked 20% honey solution was provided as adult food. Furthermore, the number of adult offspring was recorded until complete emergence. The T. dendrolimi and T. ostriniae were identified with the help of morphological descriptions provided by Myint et al. [29]. The unhatched eggs were dissected to ascertain the offspring’s mortality. All treatments were replicated ten times. Furthermore, all the emerged offspring of the F₁ generation from a single treatment were transferred to a cage containing 30 masses of ACB eggs (each mass contains 70 eggs) (aged < 4 h) stapled with the leaf surface of a corn plant. After 8 days of parasitism, 15 randomly selected parasitized egg masses were placed in a glass tube (10 × 3 cm, length × diameter; Shanghai Allcan Medical Co., Ltd., Pudong New Area, Shanghai, China) and then kept in an incubator under controlled conditions (L14: D10, 26 ± 1 °C, 65 ± 5% RH) for development. After the emergence of second generation (F₂), the species were identified based on the abovementioned procedure, and both wasps’ emergence rate and mortality were recorded. The same experimental procedure was adopted to evaluate the emergence rate and mortality of offspring of the third generation (F₃).

The offspring emergence rate (%) for T. dendrolimi and T. ostriniae was calculated based on the following formula:

$$\begin{array}{l}\mathrm{T}\mathrm{d} \mathrm{o}\mathrm{f}\mathrm{f}\mathrm{s}\mathrm{p}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}=\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d} \mathrm{T}\mathrm{d}/(\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d} \mathrm{T}\mathrm{d}\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}+\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d} \mathrm{T}\mathrm{o})\times 100\end{array}$$

$$\begin{array}{l}\mathrm{T}\mathrm{o} \mathrm{o}\mathrm{f}\mathrm{f} \mathrm{s}\mathrm{p}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}=\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d} \mathrm{T}\mathrm{o}/(\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d} \mathrm{T}\mathrm{d}\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}+\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d} \mathrm{T}\mathrm{o})\times 100\end{array}$$

The offspring mortality (%) for T. dendrolimi and T. ostriniae was calculated based on the following formula:

$$\begin{array}{l}\mathrm{T}\mathrm{d} \mathrm{o}\mathrm{f}\mathrm{f} \mathrm{s}\mathrm{p}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{m}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}=\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t} \mathrm{T}\mathrm{d}/(\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t} \mathrm{T}\mathrm{d}\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}+\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t} \mathrm{T}\mathrm{o})\times 100\end{array}$$

$$\begin{array}{l}\mathrm{T}\mathrm{o} \mathrm{o}\mathrm{f}\mathrm{f}\mathrm{s}\mathrm{p}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{m}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}=\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f}\mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t} \mathrm{T}\mathrm{o}/(\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t} \mathrm{T}\mathrm{d}\\ \hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}+\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t} \mathrm{T}\mathrm{o})\times 100\end{array}$$

2.4. Statistical Analysis

Data were analyzed by the statistical software IBM SPSS^® (Statistical Package for Social Science) v8.0. The data regarding offspring emergence and mortality were analyzed using factorial analysis of variance (ANOVA), and treatment means were further compared by Tukey’s honestly significant difference (HSD) test at a 95% confidence interval. Prior to analysis, data were normalized by arcsine square root transformation (arsin (sqrt(x))). Data were displayed using SigmaPlot 12.5.

3. Results

3.1. Effect of Parasitoid Ratio Variations on Offspring Emergence Across Generations in Trichogramma Species

The results showed that the offspring emergence rate of both Trichogramma species was significantly affected by parasitoid generations (p < 0.0001), parasitoid ratios (p < 0.0001), and parasitoid generations × parasitoid ratios (p < 0.0001) (

Table 1. The effect of parasitoid generations, parasitoid ratios, and parasitoid generations × parasitoid ratios on offspring emergence rate of both Trichogramma species from Ostrinia furnacalis eggs.

Parameter	Source	df	Trichogramma Species
			F-Value	p-Value
Offspring emergence rate (%)	Parasitoid generations	2	1101.536	<0.0001
	Parasitoid ratios	4	603.500	<0.0001
	Parasitoid generations × Parasitoid ratios	8	475.290	<0.0001
	Error	135

p < 0.001 is considered significant; two-factorial ANOVA α at = 0.05.

). In the F₁ and F₂ generations, there was a significant difference in T. dendrolimi emergence among different parasitoid ratios (F₁:F_4,45 = 23.592, p < 0.0001; F₂:F_4,45 = 4.518, p = 0.0037). Furthermore, under the parasitoid ratios of 5:1, 3:1, and 1:1, the emergence of F₁ generation of T. dendrolimi was significantly higher (27.2%, 25.7%, and 17%, respectively) than the parasitoid ratios of 1:3 (4.2%) and 1:5 (2.1%), respectively. Comparatively, in the F₂ generation, the emergence of T. dendrolimi was significantly reduced by 91.54%, 90.66%, and 93.52% under parasitoid ratios of 5:1, 3:1, and 1:1, respectively. However, no emergence of T. dendrolimi was observed under 1:3 and 1:5 ratios. Moreover, under all parasitoid ratios, no emergence of F₃ generation of T. dendrolimi was observed (Figure 1).

In the F₁ and F₂ generations, there was a significant difference in T. ostriniae emergence among different parasitoid ratios (F₁:F_4,45 = 23.592, p < 0.0001; F₂:F_4,45 = 4.518, p = 0.0037). Furthermore, under the parasitoid ratios of 1:5, and 1:3, the emergence of F₁ generation of T. ostriniae was significantly higher (97.9%, and 95.8%, respectively) than the parasitoid ratios of 1:1 (83%) and 3:1 (74.3%), and 5:1 (72.8%), respectively. Comparatively, in the F₂ generation, the emergence of T. ostriniae was significantly increased by 2.14%, and 4.38%, 19.15%, 31.35% and 34.20% under parasitoid ratios of 1:5, 1:3, 1:1, 3:1 and 5:1, respectively. Moreover, under all parasitoid ratios, complete emergence of F₃ generation of T. ostriniae was observed (Figure 1).

Under the same parasitoid ratios, we compared the T. dendrolimi emergence among different generations and found significant differences (5:1:F_2,27 = 92.828, p < 0.0001, 3:1:F_2,27 = 50.765, p < 0.0001, 1:1:F_2,27 = 76.300, p < 0.0001, 1:3:F_2,27 = 11.780, p = 0.0002), indicating that as the number of generations increased, the offspring emergence was gradually decreased. However, under the 1:5 parasitoid ratios, there is no significant difference among various generations for the T. dendrolimi emergence (F_2,27 = 3.724, p = 0.0563). The complete disappearance of T. dendrolimi progeny was exhibited by an F₃ generation in 5:1, 3:1, and 1:1 ratio, whereas under 1:3 and 1:5 ratios, no emergence of T. dendrolimi progeny occurred in the F₂ generation (Figure 1).

Similarly, under the same parasitoid ratios, we compared the T. ostriniae emergence among different generations and found significant differences (5:1:F_2,27 = 92.828, p < 0.0001, 3:1:F_2,27 = 50.765, p < 0.0001, 1:1:F_2,27 = 76.300, p < 0.0001, 1:3:F_2,27 = 11.780, p = 0.0002). However, unlike T. dendrolimi, a contradictory trend was observed, indicating that offspring emergence significantly increased as the number of generations increased. Nevertheless, under the 1:5 parasitoid ratios, there is no significant difference among various generations for the T. ostriniae emergence (F_2,27 = 3.724, p = 0.0563). The F₃ generation exhibited a complete emergence of T. ostriniae under the 5:1, 3:1, and 1:1 parasitoid ratios, whereas under 1:3 and 1:5 ratios, complete emergence of T. ostriniae progeny occurred in the F₂ generation (Figure 1). Regardless of the parasitoid ratios, the offspring emergence of T. ostriniae in all three generations was significantly higher than that of T. dendrolimi (All p < 0.05) (Figure 1).

3.2. Effect of Parasitoid Ratio Variations on Offspring Mortality Across Generations in Trichogramma Species

After assessing the offspring mortality in our research by dissecting the unhatched eggs, we found that (based on ANOVA) the offspring mortality in both Trichogramma species was significantly affected by parasitoid generations (p < 0.0001) but was not significantly affected by parasitoid ratios (p = 0.3855), or interaction between parasitoid generations and parasitoid ratios (p = 0.9050) (

Table 2. The effect of parasitoid generations, parasitoid ratios, and parasitoid generations × parasitoid ratios on offspring mortality of both Trichogramma species from Ostrinia furnacalis eggs.

Parameter	Source	df	Trichogramma Species
			F-Value	p-Value
Offspring mortality (%)	Parasitoid generations	2	25.609	<0.0001
	Parasitoid ratios	4	1.047	0.3855
	Parasitoid generations × Parasitoid ratios	8	0.424	0.9050
	Error	135

p < 0.001 is considered significant; two-factorial ANOVA α at = 0.05.

). In the F₁, F₂, and F₃ generations, there was not a significant difference in T. dendrolimi mortality among different parasitoid ratios (F₁:F_4,42 = 0.495, p = 0.7392; F₂:F_4,42 = 3.882, p = 0.0881, F₃:F_4,42 = 3.882, p = 0.0881). The same trend was observed for offspring mortality of T. ostriniae in all three generations (F₁:F_4,42 = 0.495, p = 0.7392; F₂:F_4,42 = 3.882, p = 0.0881, F₃:F_4,42 = 3.882, p = 0.0881). Furthermore, under the parasitoid ratios of 1:3, and 1:5, there was no existence of T. dendrolimi in F₂ and F₃ generations (Figure 2).

Under the 5:1, 3:1, 1:1, and 1:3 parasitoid ratios, we compared the T. dendrolimi mortality among different generations and found significant differences (5:1:F_2,25 = 5.358, p = 0.0116, 3:1:F_2,27 = 8.229, p = 0.0016, 1:1:F_2,27 = 3.636, p = 0.0400, 1:3:F_2,26 = 4.416, p = 0.0223), indicating that offspring mortality significantly decreased as the number of generations increased. Additionally, no significant difference was observed between F₁ and F₂; however, a significant difference was noted in the F₃ generation regarding T. dendrolimi mortality under the 5:1, 3:1, and 1:1 parasitoid ratios. In the 1:3 and 1:5 ratios, the T. dendrolimi mortality in the F₁ generation was higher than F₂ and F₃ generations (Figure 2).

Similarly, significant differences were observed in T. ostriniae mortality under the parasitoid ratios 5:1, 3:1, 1:1, and 1:3 (5: 1:F_2,25 = 5.358, p = 0.0116, 3: 1:F_2,27 = 8.229, p = 0.0016, 1:1:F_2,27 = 3.636, p = 0.0400, 1:3:F_2,26 = 4.416, p = 0.0223). Though, dissimilar to T. dendrolimi, a contrary trend was observed, indicating that offspring mortality of T. ostriniae significantly increased as the number of generations increased. Also, when the parasitoid ratios were 5:1, 3:1, and 1:1, there was no significant difference in the offspring mortality of T. ostriniae between the F₁ and F₂ generations, but there was a considerable difference compared to the F₃ generation. In the 1:3 and 1:5 ratios, the mortality of T. ostriniae offspring in the F₁ generation is higher than in the F₂ and F₃ generations. Overall, the mortality of T. ostriniae offspring was significantly higher than that of T. dendrolimi (All p < 0.05) in all parasitoid ratios and generations (Figure 2).

4. Discussion

Parasitoids are considered long-term, cost-effective and sustainable solutions to manage insect pests in agriculture [30]. Studies on the performance of parasitoid T. ostriniae on ACB eggs and investigating the offspring’s emergence through multiparasitism along with T. dendrolimi are needed. To better understand the dynamics of multiparasitism, reproductive success plays a key role in analyzing the effectiveness of Trichogramma in a biological control program [25]. Our research found significant reproductive success of T. ostriniae over T. dendrolimi. Furthermore, the offspring emergence and mortality of two Trichogramma parasitoid species (T. dendrolimi and T. ostriniae) significantly differed across multiple generations based on parasitoid ratios for the ACB host. The combine and multiple release of two Trichogramma species is a promising biocontrol strategy to control insect pests in the field [31,32]. Sigsgaard et al. [33] evaluated the mix of two Trichogramma species, T. evanescens and T. cacoeciae (1:1), to control Cydia pomonella in apple orchards and revealed a 54% reduction in fruit damage in the fields. However, the scientists did not identify the most promising and potential biocontrol candidate between T. evanescens and T. cacoeciae to control C. pomonella eggs. Moreover, they only utilized the same ratio of both Trichogramma species. However, in our research, we used various ratios of T. dendrolimi and T. ostriniae to parasitize ACB eggs and estimated the offspring emergence and mortality across three generations. The results indicated that the highest emergence of the F₁ generation of T. dendrolimi was observed when five T. dendrolimi and one T. ostriniae were reared on ACB eggs. Apart from the higher density of T. dendrolimi (five female parasitoids) concerning T. ostriniae (a single female parasitoid), T. dendrolimi had better temperature and humidity adaptation than other Trichogrmma species, making T. dendrolimi a potential candidate for controlling insect pests [34]. Unfortunately, T. ostriniae put an evil eye on the reproductive success of T. dendrolimi because, at the same parasitoid ratios (5:1) as the generation increase from F₁ to F₂, and F₂ to F₃, the offspring emergence of T. dendrolimi was reduced by 91.54% and 100%, respectively. The T. ostriniae is accountable for this sudden reduction in T. dendrolimi offspring emergence because the former efficiently parasitized the ACB eggs irrespective of host age, whereas older host eggs are unsuitable for T. dendrolimi [35,36]. Moreover, T. ostriniae prefers to oviposit the yellow host (ACB egg) compared to T. dendrolimi [37]. Additionally, parasitoids may cause egg mortality by multiple drilling without laying eggs in the host [38]. Therefore, the offspring emergence of T. dendrolimi was gradually suppressed. Furthermore, the highest emergence of the F₁ generation of T. ostriniae was observed when one T. dendrolimi and five T. ostriniae were reared on ACB eggs. Therefore, due to its higher density, T. ostriniae benefited from T. dendrolimi during interspecific competition [39]. Additionally, T. ostriniae developed significantly faster at every age of ACB eggs than T. dendrolimi [35]. Therefore, T. ostriniae outperformed T. dendrolimi [40]. Finally, the emergence of T. ostriniae progeny increased with the number of generations. Thus, the population growth rate will serve as an adequate indicator of parasitoid performance on different hosts [41]. In addition to progeny emergence, understanding parasitoid mortality is crucial for successful insect pest management [42]. Additionally, mortality is arguably the most widespread empirical measure in entomology [43]. After assessing the offspring mortality in our research by dissecting the unhatched eggs, we found that, across all parasitoid ratios and generations, the offspring mortality of T. ostriniae was considerably greater than that of T. dendrolimi (All p < 0.05). These results suggest that mortality is a crucial empirical measure that validates T. ostriniae’s superiority over T. dendrolimi. Moreover, Wang et al. [35] also revealed that T. ostriniae is more effective than T. dendrolimi as a biocontrol agent of the ACB eggs. These findings highlight the importance of selecting suitable parasitoid species when implementing Trichogramma for pest management. Further research should build upon these semi-field observations by examining conditions where additional ecological factors may influence parasitoid competition. A comprehensive understanding of parasitoid interactions during multi-generational rearing on ACB eggs will enhance the effectiveness of biological control programs targeting lepidopteran pests.

5. Conclusions

The research results indicate that parasitoid generations and ratios affect the emergence and mortality of T. dendrolimi and T. ostriniae offspring. Due to the complete emergence of T. ostriniae in the F₃ generations, T. ostriniae outperformed T. dendrolimi regardless of the parasitoid ratios. Additionally, after dissecting the unhatched eggs, the dead offspring was detected. We found that the progeny mortality of T. ostriniae was significantly higher than that of T. dendrolimi across all parasitoid ratios and generations, indicating that T. ostriniae was more prevalent than T. dendrolimi. According to these findings, T. ostriniae performs the best and is the most viable option for biologically controlling ACB in the fields, eventually increasing maize productivity.